Anti-RPE65 antibody [EPR22579-44]

9 product images

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  Immunoprecipitation - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  RPE65 was immunoprecipitated from 0.35 mg of mouse eyeball lysate with ab231782 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab231782 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used as secondary antibody at 1/5000 dilution.
  

  Lane 1: Mouse eyeball lysate 10 μg (Input).
  Lane 2: ab231782 IP in mouse eyeball lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab231782 in mouse eyeball lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  Exposure time: 3 minutes.

  All lanes: Immunoprecipitation - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Predicted band size: 60 kDa

  Observed band size: 65 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Immunohistochemical analysis of paraffin-embedded mouse retina tissue labeling RPE65 with ab231782 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on pigment epithelium cells of mouse retina (PMID: 25941382, PMID: 22238088 is observed. Counter stained with hematoxylin.
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
  

  Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Immunohistochemistry (Frozen sections) - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Immunohistochemical analysis of frozen section of 4%PFA-fixed, 0.2% Triton X-100 permeabilized rat retinal tissue labeling RPE65 with ab231782 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution (green). Positive staining on retinal pigment epithelium (PMID: 17848510) is observed. The nuclear counter stain is DAPI (blue).
  

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

  Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

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  Western blot - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Blocking and dilution buffer: 5% NFDM/TBST.
  

  Exposure times.

  Lane 1: 26 seconds. Lanes 2 & 3: 3 minutes.

  The molecular weight observed is consistent with what has been described in the literature (PMID:17848510).

  All lanes: Western blot - Anti-RPE65 antibody [EPR22579-44] (ab231782) at 1/1000 dilution

  Lane 1: Human eye tissue lysate at 20 µg

  Lane 2: Mouse eye tissue lysate at 20 µg

  Lane 3: Rat eye tissue 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: 60 kDa

  Observed band size: 65 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Immunohistochemical analysis of paraffin-embedded human retina tissue labeling RPE65 with ab231782 at 1/4000 dilution, followed by ready to use AP-labeled secondary antibody kit. Cytoplasmic staining on pigment epithelium cells of human retina (PMID: 25941382; PMID: 22238088) is observed. Counter stained with hematoxylin.
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use AP-labeled secondary antibody kit.
  

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  The section was incubated with ab231782 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• 

  Immunohistochemistry (Frozen sections) - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Immunohistochemical analysis of frozen section of 4%PFA-fixed, 0.2% Triton X-100 permeabilized mouse retinal tissue labeling RPE65 with ab231782 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution (green). Positive staining on retinal pigment epithelium (PMID: 17848510) is observed. The nuclear counter stain is DAPI (blue).
  

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

  Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Immunohistochemical analysis of paraffin-embedded rat retina tissue labeling RPE65 with ab231782 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on pigment epithelium cells of rat retina (PMID: 25941382, PMID: 22238088) is observed. Counter stained with hematoxylin.
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
  

  Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Western blot - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  False colour image of Western blot: Anti-RPE65 antibody [EPR22579-44] 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, ab231782 was shown to bind specifically to RPE65. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-RPE65 antibody [EPR22579-44] (ab231782) at 1/1000 dilution

  Lane 1: Human Eye cell lysate at 20 µg

  Lane 2: Mouse Eye cell lysate at 20 µg

  Lane 3: Rat Eye cell lysate at 20 µg

  Lane 4: Y79 cell lysate at 20 µg

  Lane 5: PC-3 cell lysate at 20 µg

  Performed under reducing conditions.

  Observed band size: 65 kDa

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  Multiplex immunohistochemistry - Anti-RPE65 antibody [EPR22579-44] (ab231782)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human retina tissue labeling RPE65, Rhodopsin and Synapsin I with ab231782 at 1/8000 dilution, Anti-Rhodopsin antibody [EPR21876] - BSA and Azide free ab232934 at 1/8000 dilution and Anti-Synapsin I antibody [EPR23531-50] - BSA and Azide free ab274430 at 1/1500 dilution followed by a ready to use Opal Polymer HRP Ms + Rb secondary antibody. Nuclear counter stain used was DAPI.
  
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
  
  Panel A: merged staining of anti-Synapsin I (magenta; Opal™690), anti-RPE65 (green; Opal™520) and anti-Rhodopsin (red; Opal™570) on human retina.
  Panel B: anti-RPE65 stained on pigmented layer.
  Panel C: anti-Rhodopsin stained on rod photoreceptor cells.
  Panel D: anti-Synapsin I stained on inner plexiform layer.
  
  The section was incubated in three rounds of staining: in the order of Anti-Synapsin I antibody [EPR23531-50] - BSA and Azide free ab274430, ab231782, and Anti-Rhodopsin antibody [EPR21876] - BSA and Azide free ab232934 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.
  
  The section was incubated in three rounds of staining: in the order of Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840, Anti-CD3 epsilon antibody [SP7] ab16669, and Anti-CD20 antibody [SP32] - BSA and Azide free ab236434 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.