Anti-TMEM119 antibody [28-3] - Microglial marker

• IHC-Fr

AbReview59215****

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

ab209064 staining TMEM119 in Mouse corpus callosum sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 0.5% BSA for 1 hour at 23°C. Samples were incubated with primary antibody at 1.4μg/ml for 18 hours at 4°C. An Alexa Fluor® 488 -conjugated Donkey anti-rabbit IgG polyclonal was used as the secondary antibody.

TMEM119 (green), Iba1 (red) and DAPI (blue)

This image is courtesy of an anonymous Abreview

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 staining in a section of frozen normal mouse brain wild type (upper panel) and TMEM119 knockout (lower panel). No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in PBS containing 0.5% (v/v) Triton X-100, 0.3 M (w/v) glycine and 1% (w/v) BSA for 1 h at room temperature. The section was then incubated overnight at +4°C in PBS containing 0.5% (v/v) Triton X-100 and 1% (w/v) BSA with ab209064 at 0.5 μg/ml. The secondary antibody was ab150087 (shown in red) used at 2 μg/ml for 1 hour at room temperature. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount®. Images were taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Normal (WT) mouse brain, stained for TMEM119 (red), Iba1 (green) and DAPI (blue). Samples were baked onto slides for 10 minutes at 60^oC, rehydrated with PBS and blocked with blocking buffer (10% serum in PBST). ab209064 at a concentration of 1 μg/mL was incubated with the sample overnight at 4^oC. Slides were washed with PBS and a goat anti-rabbit Alexa Fluor 488^® was used as the secondary antibody at a concentration of 4 μg/mL.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

ab209064 at 1 : 2000 staining TMEM119 antibody in mouse cerebrum tissue by immunohistochemistry (FFPE). Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling TMEM119 with ab209064 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP). Positive staining on glial cells in mouse cerebrum is observed. Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0) before commencing with IHC staining protocol. Counter stained with hematoxylin.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 staining in a section of frozen normal mouse brain. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in PBS containing 0.5% (v/v) Triton X-100, 0.3 M (w/v) glycine and 1% (w/v) BSA for 1 h at room temperature. The section was then incubated overnight at +4°C in PBS containing 0.5% (v/v) Triton X-100 and 1% (w/v) BSA with ab209064 at 0.5 μg/ml. The secondary antibody was ab150087 (shown in red) used at 2 μg/ml for 1 hour at room temperature. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 and Iba1 co-staining in a section of formalin-fixed paraffin-embedded normal mouse brain. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked in PBS containing 0.5% (v/v) Triton X-100, 0.3 M (w/v) glycine and 1% (w/v) BSA for 1 h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.5% (v/v) Triton X-100 and 1% (w/v) BSA with ab209064 at 1 μg/ml and ab5076 at 5 μg/ml. The secondary antibodies were ab150087 (shown in red) and ab150133 (shown in green) used at 2 μg/ml for 1 hour at room temperature. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount®. Images were taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Normal mouse choroid plexus, stained for TMEM119 (red), Iba1 (green) and DAPI (blue). Choroid plexus macrophages are positive for Iba1 and negative for TMEM119. Samples were baked onto slides for 10 minutes at 60^oC, rehydrated with PBS and blocked with blocking buffer (10% serum in PBST). ab209064 at a concentration of 1 μg/mL was incubated with the sample overnight at 4^oC. Slides were washed with PBS and a goat anti-rabbit Alexa Fluor 488^® was used as the secondary antibody at a concentration of 4 μg/mL.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 staining in a section of formalin fixed, paraffin embedded normal mouse brain. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked in PBS containing 0.5% (v/v) Triton X-100, 0.3 M (w/v) glycine and 1% (w/v) BSA for 1 h at room temperature. The section was then incubated overnight at +4°C in PBS containing 0.5% (v/v) Triton X-100 and 1% (w/v) BSA with ab209064 at 0.1 μg/ml. The secondary antibody was ab150087 (shown in red) used at 2 μg/ml for 1 hour at room temperature. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount®. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-Fr

PubMed

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Representative FISH analysis of GAS5 (green) co‐stained with ab209064 (red) in spinal cord sections from EAE mice at 30 dpi. Arrows indicate GAS5⁺TMEM119⁺ cells. Scale bars = 25 μm.

Female C57BL/6 mice (6–8 weeks) were deeply anesthetized with 3% chloral hydrate and a laminectomy was performed. After fixing the spine, 1 μl of 1% lysolecithin in a 0.9% sodium chloride solution was injected into the dorsal funiculus at the level of the T11–T12 vertebrae. The day of lysolecithin injection was designated day 0 (0 dpi). The spinal cord around the injection point was isolated and cut into serial cryosections.

Tissue sections were fixed, permeabilized, and incubated with the primary antibody overnight at 4°C, followed by 2 h of incubation with TRITC‐ or FITC‐conjugated secondary antibodies. Then, the samples were counterstained with Hoechst 33342.

Image from Sun D et al., EMBO Rep.. 2017;18(10):1801-1816. Fig EV3.; doi: 10.15252/embr.201643668. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• IHC-FoFr

Lab

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 staining in a section of Mouse cerebrum using ab209064 at 1 : 50 dilution.The section was fixed with 4% PFA then permeabilized with 0.2% Triton X-100. The secondary antibody was ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed used at 1 : 1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Secondary antibody only control : PBS instead of the primary antibody.

Positive staining on mouse cerebrum.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

IHC image of TMEM119 staining in a section of formalin fixed, paraffin embedded normal mouse brain, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab209064, 0.5 μg/ml, for 15 mins at room temperature. A goat anti-Rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• IHC-Fr

PubMed

Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Representative images of sham (d–g) and hypoperfusion (h–k) at 12 weeks post-surgery are shown to illustrate Iba-1 immunostaining in sham (d) and hypoperfused (h); TMEM119 immunostaining in sham (e) and hypoperfused (i) and then Iba-1/TMEM119 co-localisation in sham (f,g) and hypoperfused (j,k) white matter. All Iba-1⁺ cells in both sham and hypoperfused cohorts were also TMEM119⁺ indicating that the cells in the corpus callosum were resident microglia. Scale bars; d-f and h-j are 50μm, g and k 10 μm. The number of microglial cells significantly correlated with nodal gap length.

Free floating cryo-preserved sections cut at 30 μm thickness. Sections were incubated with the primary antibodies (anti-Iba-1 (1/100) and anti-TMEM119 (1/500, ab209064)) overnight at 4°C. Sections were stained at the outset with haematoxylin and eosin to determine the presence and absence of ischemic neuronal perikaryal damage as part of the inclusion/exclusion criteria.

Image from Manso Y et al., Glia. 2018;66(1):34-46. Fig 4.; doi: 10.1002/glia.23190 Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse cerebrum labelling TMEM119 with ab209064 at 1/1000 dilution. The secondary used was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Sections were counterstained with DAPI. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Positive staining on microglial cells of mouse cerebrum. The section was incubated with ab209064 at 4°C overnight.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (AB209064)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse brain labelling TMEM119 with ab209064 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab209064 anti-TMEM119 antibody [28-3] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).