Anti-FMRP antibody [EPR23852-90]

33 product images

• 

  Immunoprecipitation - Anti-FMRP antibody [EPR23852-90] (ab259335)

  FMRP was immunoprecipitated from 0.35 mg C2C12 (Mouse myoblast) whole cell lysate 10ug with ab259335 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259335 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  

  Lane 1: C2C12 (Mouse myoblast) whole cell lysate 10ug

  Lane 2: ab259335 IP in C2C12 whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab259335 in C2C12 whole cell lysate

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 10 seconds.

  All lanes: Immunoprecipitation - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Predicted band size: 71 kDa

  Observed band size: 80 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on mouse cerebrum (PMID: 24463622). The section was incubated with ab259335 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• 

  Flow Cytometry (Intracellular) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized C2C12 (Mouse myoblast) cells labelling FMRP with ab259335 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
  

  A Goat anti rabbit IgG (Alexa Fluor^®488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

• 

  Immunoprecipitation - Anti-FMRP antibody [EPR23852-90] (ab259335)

  FMRP was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell ) whole cell lysate with ab259335 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259335 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  

  Lane 1: HeLa (human cervix adenocarcinoma epithelial cell ) whole cell lysate 10ug

  Lane 2: ab259335 IP in HeLa whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab259335 in HeLa whole cell lysate

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 10 seconds.

  All lanes: Immunoprecipitation - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Predicted band size: 71 kDa

  Observed band size: 80 kDa

• 

  Immunohistochemistry (Frozen sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  IHC image of ab259335 staining FMRP in rat testis frozen tissue sections, performed on a Leica Biosystems BOND® RX instrument. The section was incubated with ab259335 at 0.5μg/ml for 30 mins at room temperature and detected using an HRP conjugated compact polymer system (Bond™ Polymer Refine Detection). DAB was used as the chromogen. Positive staining was seen in cytoplasm and membrane in rat testis. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  

  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C2C12 cells labelling FMRP with ab259335 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic and weak nuclear staining in C2C12 cells. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• 

  Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Lanes 1 - 4: Merged signal (red and green). Green - ab259335 observed at 77 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
  

  ab259335 was shown to react with FMRP in western blot. The band observed in the CRISPR/Cas9 edited lysate lane below 77 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab259335 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: FMRP CRISPR/Cas9 edited HAP1 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Human brain cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 71 kDa

  Observed band size: 77 kDa

• 

  Immunohistochemistry (Frozen sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  IHC image of ab259335 staining FMRP in mouse testis frozen tissue sections, performed on a Leica Biosystems BOND® RX instrument. The section was incubated with ab259335 at 0.5μg/ml for 30 mins at room temperature and detected using an HRP conjugated compact polymer system (Bond™ Polymer Refine Detection). DAB was used as the chromogen. Positive staining was seen in cytoplasm and membrane in mouse testis. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  

  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Blocking and dilution buffer: 5% NFDM/TBST.
  

  Exposure time: 81 seconds.

  Negative control: Mouse heart(PMID: 7633436).

  Multiple bands could be seen due to alternative splicing of FMRP

  All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate at 20 µg

  Lane 2: Mouse heart tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 71 kDa

  Observed band size: 75 kDa, 80 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on rat testis. The section was incubated with ab259335 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• 

  Immunohistochemistry (Frozen sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  IHC image of ab259335 staining FMRP in rat brain frozen tissue sections, performed on a Leica Biosystems BOND® RX instrument. The section was incubated with ab259335 at 0.5μg/ml for 30 mins at room temperature and detected using an HRP conjugated compact polymer system (Bond™ Polymer Refine Detection). DAB was used as the chromogen. Positive staining was seen in cytoplasm and membrane in rat brain. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  

  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  Multiple bands could be seen due to alternative splicing of FMRP.
  Exposure time: 180 seconds.

  All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate at 20 µg

  Lane 2: Mouse hippocampus tissue lysate at 20 µg

  Lane 3: Rat hippocampus tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 71 kDa

  Observed band size: 75 kDa, 80 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on rat cerebrum. The section was incubated with ab259335 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on mouse testis (PMID: 16790844). The section was incubated with ab259335 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• 

  Immunohistochemistry (Frozen sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  IHC image of ab259335 staining FMRP in mouse brain frozen tissue sections, performed on a Leica Biosystems BOND® RX instrument. The section was incubated with ab259335 at 0.5μg/ml for 30 mins at room temperature and detected using an HRP conjugated compact polymer system (Bond™ Polymer Refine Detection). DAB was used as the chromogen. Positive staining was seen in cytoplasm and membrane in mouse brain. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  

  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  Exposure time: 26 seconds.

  All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

  Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

  Lane 3: PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 71 kDa

  Observed band size: 80 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Negative control: No staining on mouse cardiac muscle (PMID: 7633436). The section was incubated with ab259335 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• 

  Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335)

  False colour image of Western blot: Anti-FMRP antibody [EPR23852-90] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab259335 was shown to bind specifically to FMRP. A band was observed at 70-77 kDa in wild-type A549 cell lysates with no signal observed at this size in Fmr1 knockout cell line Human FMR1 knockout A549 cell line ab288956. To generate this image, wild-type and Fmr1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: Fmr1 knockout A549 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Human Brain cell lysate at 20 µg

  Performed under reducing conditions.

  Observed band size: 70-77 kDa

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse cerebrum tissue sections.
  

  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on mouse cerebrum.

  Panel B: anti-GPR17 staining oligodendrocytes in mouse cerebrum.

  Panel C: anti-P2RY12 staining microglia in mouse cerebrum.

  Panel D: anti-FMR1 staining neurons in mouse cerebrum.

  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse hippocampus tissue sections.
  

  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on mouse hippocampus.

  Panel B: anti-GPR17 staining oligodendrocytes in mouse hippocampus.

  Panel C: anti-P2RY12 staining microglia in mouse hippocampus.

  Panel D: anti-FMR1 staining neurons in mouse hippocampus.

  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Nuclear counterstaining with DAPI.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded rat cerebrum tissue sections.

  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on rat cerebrum.

  Panel B: anti-GPR17 staining oligodendrocytes in rat cerebrum.

  Panel C: anti-P2RY12 staining microglia in rat cerebrum.

  Panel D: anti-FMR1 staining neurons in rat cerebrum.

  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Nuclear counterstaining with DAPI.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded rat hippocampus tissue sections.

  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on rat hippocampus.

  Panel B: anti-GPR17 staining oligodendrocytes in rat hippocampus.

  Panel C: anti-P2RY12 staining microglia in rat hippocampus.

  Panel D: anti-FMR1 staining neurons in rat hippocampus.

  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Nuclear counterstaining with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse spinal cord tissue.

  
  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on mouse spinal cord.
  
  Panel B: anti-GPR17 staining oligodendrocytes in mouse spinal cord.
  
  Panel C: anti-P2RY12 staining microglia in mouse spinal cord.
  
  Panel D: anti-FMR1 staining neurons in mouse spinal cord.
  
  Nuclear DNA was labeled with DAPI (shown in blue).
  

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebellum labelling Noradrenaline transporter with Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361 at 1/100 (B), GPCR GPR17 with Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 at 1/2000 dilution (C) and FMRP with ab259335 at 1/10000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  
  Panel A: merged staining of anti-Noradrenaline transporter (magenta; Opal™690), anti-GPCR GPR17 (green; Opal™520) and anti-FMRP (gray; Opal™570) on mouse cerebellum.
  
  Panel B: anti-Noradrenaline transporter staining nerves in mouse cerebellum.
  
  Panel C: anti-GPCR GPR17 staining oligodendrocytes in mouse cerebellum.
  
  Panel D: anti-FMRP staining neurons in mouse cerebellum.
  

  
  The section was incubated in three rounds of staining: in the order of Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361, Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 and ab259335 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
  

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse cerebrum tissue staining TRIM46 with Anti-TRIM46 antibody [EPR26957-34] ab307967 at a 1:5000 (0.102 ug/ml) dilution, FMRP with ab259335 at 1:10000 (0.04 ug/ml) dilution and Serotonin transporter with Anti-Serotonin transporter antibody [EPR26259-61] ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

  Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-FMRP (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on mouse cerebrum.
  Panel B: anti-TRIM46 staining the proximal part of the axon in mouse cerebrum.
  Panel C: anti-FMRP staining neurons in mouse cerebrum.
  Panel D: anti-Serotonin transporter staining dendrites in mouse cerebrum.
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-TRIM46 antibody [EPR26957-34] ab307967, ab259335 and Anti-Serotonin transporter antibody [EPR26259-61] ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse hippocampus tissue staining TRIM46 with Anti-TRIM46 antibody [EPR26957-34] ab307967 at a 1:5000 (0.102 ug/ml) dilution, FMRP with ab259335 at 1:10000 ( 0.04 µg/ml) dilution and Serotonin transporter with Anti-Serotonin transporter antibody [EPR26259-61] ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

  Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-FMRP (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on mouse hippocampus.
  Panel B: anti-TRIM46 staining the proximal part of the axon in mouse hippocampus.
  Panel C: anti-FMRP staining neurons in mouse hippocampus.
  Panel D: anti-Serotonin transporter staining dendrites in mouse hippocampus.
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-TRIM46 antibody [EPR26957-34] ab307967, ab259335 and Anti-Serotonin transporter antibody [EPR26259-61] ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat cerebrum tissue staining TRIM46 with Anti-TRIM46 antibody [EPR26957-34] ab307967 at a 1:5000 (0.102 ug/ml) dilution, FMRP with ab259335 at 1:10000 (0.04 ug/ml) dilution and Serotonin transporter with Anti-Serotonin transporter antibody [EPR26259-61] ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

  Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-FMRP (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on rat cerebrum.
  Panel B: anti-TRIM46 staining the proximal part of the axon in rat cerebrum.
  Panel C: anti-FMRP staining neurons in rat cerebrum.
  Panel D: anti-Serotonin transporter staining dendrites in rat cerebrum.
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-TRIM46 antibody [EPR26957-34] ab307967, ab259335 and Anti-Serotonin transporter antibody [EPR26259-61] ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat hippocampus tissue staining TRIM46 with Anti-TRIM46 antibody [EPR26957-34] ab307967 at a 1:5000 (0.102 ug/ml) dilution, FMRP with ab259335 at 1:10000 (0.04 ug/ml) dilution and Serotonin transporter with Anti-Serotonin transporter antibody [EPR26259-61] ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

  Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-FMRP (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on rat hippocampus.
  Panel B: anti-TRIM46 staining the proximal part of the axon in rat hippocampus.
  Panel C: anti-FMRP staining neurons in rat hippocampus.
  Panel D: anti-Serotonin transporter staining dendrites in rat hippocampus..
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-TRIM46 antibody [EPR26957-34] ab307967, ab259335 and Anti-Serotonin transporter antibody [EPR26259-61] ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat spinal cord tissue staining TRIM46 with Anti-TRIM46 antibody [EPR26957-34] ab307967 at a 1:5000 (0.102 ug/ml) dilution, FMRP with ab259335 at 1:10000 (0.04 ug/ml) dilution and Serotonin transporter with Anti-Serotonin transporter antibody [EPR26259-61] ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

  Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-FMRP (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on rat spinal cord.
  Panel B: anti-TRIM46 staining the proximal part of the axon in rat spinal cord.
  Panel C: anti-FMRP staining neurons in rat spinal cord.
  Panel D: anti-Serotonin transporter staining dendrites in rat spinal cord.
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-TRIM46 antibody [EPR26957-34] ab307967, ab259335 and Anti-Serotonin transporter antibody [EPR26259-61] ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebellum labelling Noradrenaline transporter with Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361 at 1/100 (B), GPCR GPR17 with Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 at 1/2000 dilution (C) and FMRP with ab259335 at 1/10000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  
  Panel A: merged staining of anti-Noradrenaline transporter (magenta; Opal™690), anti-GPCR GPR17 (green; Opal™520) and anti-FMRP (gray; Opal™570) on rat cerebellum.
  
  Panel B: anti-Noradrenaline transporter staining nerves in rat cerebellum.
  
  Panel C: anti-GPCR GPR17 staining oligodendrocytes in rat cerebellum.
  
  Panel D: anti-FMRP staining neurons in rat cerebellum.
  

  
  The section was incubated in three rounds of staining: in the order of Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361, Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 and ab259335 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
  

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum labelling Noradrenaline transporter with Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361 at 1/100 (B), GPCR GPR17 with Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 at 1/2000 dilution (C) and FMRP with ab259335 at 1/10000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  
  Panel A: merged staining of anti-Noradrenaline transporter (magenta; Opal™690), anti-GPCR GPR17 (green; Opal™520) and anti-FMRP (gray; Opal™570) on rat cerebrum.
  
  Panel B: anti-Noradrenaline transporter staining nerves in rat cerebrum.
  
  Panel C: anti-GPCR GPR17 staining oligodendrocytes in rat cerebrum.
  
  Panel D: anti-FMRP staining neurons in rat cerebrum.
  

  
  The section was incubated in three rounds of staining: in the order of Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361, Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 and ab259335 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
  

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum labelling Noradrenaline transporter with Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361 at 1/100 (B), GPCR GPR17 with Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 at 1/2000 dilution (C) and FMRP with ab259335 at 1/10000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  
  Panel A: merged staining of anti-Noradrenaline transporter (magenta; Opal™690), anti-GPCR GPR17 (green; Opal™520) and anti-FMRP (gray; Opal™570) on mouse cerebrum.
  
  Panel B: anti-Noradrenaline transporter staining nerves in mouse cerebrum.
  
  Panel C: anti-GPCR GPR17 staining oligodendrocytes in mouse cerebrum.
  
  Panel D: anti-FMRP staining neurons in mouse cerebrum.
  

  
  The section was incubated in three rounds of staining: in the order of Anti-Noradrenaline transporter antibody [EPR23527-534] ab254361, Anti-GPCR GPR17 antibody [EPR26422-118] ab314307 and ab259335 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
  

• 

  Multiplex immunohistochemistry - Anti-FMRP antibody [EPR23852-90] (ab259335)

  Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse cerebellum tissue sections.

  Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-FMR1 (gray; Opal™570) on mouse cerebellum.

  Panel B: anti-GPR17 staining oligodendrocytes in mouse cerebellum.

  Panel C: anti-P2RY12 staining microglia in mouse cerebellum.

  Panel D: anti-FMR1 staining neurons and Purkinje cells in mouse cerebellum.

  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution and ab259335 at a 1/10000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.