Anti-RPE65 antibody [EPR22579-44]

• mIHC

Lab

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, ab232934 at 1/8000 dilution and 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 ab274430, ab231782, and 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 ab312840, ab16669, and 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.

• IHC-P

Supplier Data

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.

• IHC-Fr

Supplier Data

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) (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) (ab150077) at 1/1000 dilution.

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

• IHC-P

Supplier Data

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 ab93684 (Tris/EDTA buffer, pH 9.0).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-RPE65 antibody [EPR22579-44] (AB231782)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse retina tissue staining RPE65 with ab231782 at a 1/8000 dilution, ab109233 anti-PAX6 used at 1/1000 dilution and ab221664 anti-RHO used at a 1/30000 dilution.

Panel A : merged staining of anti-RPE65 (green; Opal™520), anti-PAX6 (magenta; Opal™690) and anti-RHO (gray; Opal™570) on mouse retina.

Panel B : anti-RPE65 staining pigmented layer in mouse retina.

Panel C : ant-PAX6 staining inner nuclear layer and ganglion cell layer in mouse retina.

Panel D : ant-RHO staining rod-shaped photoreceptor cells in mouse retina.

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

The section was incubated in three rounds of staining : in the order of ab231782, ab109233 and ab221664 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-RPE65 antibody [EPR22579-44] (AB231782)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse retina tissue staining RPE65 with ab231782 at a 1/8000 dilution, ab308463 anti-CPLX3 used at 1/5000 dilution and ab303504 anti-ABCA4 used at a 1/500 dilution.

Panel A : merged staining of anti-RPE65 (green; Opal™520), anti-CPLX3 (magenta; Opal™690) and anti-ABCA4 (gray; Opal™570) on mouse retina.
Panel B : anti-RPE65 staining pigmented layer in mouse retina.
Panel C : ant-CPLX3 staining ribbon synapses of photoreceptors and bipolar cells in mouse retina.
Panel D : ant-ABCA4 staining photoreceptor outer segments in mouse retina.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab231782, ab308463 and ab303504 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-Fr

Supplier Data

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) (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) (ab150077) at 1/1000 dilution.

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

• IHC-P

Supplier Data

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 ab93684 (Tris/EDTA buffer, pH 9.0).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-RPE65 antibody [EPR22579-44] (AB231782)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat retina tissue staining RPE65 with ab231782 at a 1/8000 dilution, ab308463 anti-CPLX3 used at 1/5000 dilution and ab303504 anti-ABCA4 used at a 1/500 dilution.

Panel A : merged staining of anti-RPE65 (green; Opal™520), anti-CPLX3 (magenta; Opal™690) and anti-ABCA4 (gray; Opal™570) on rat retina.
Panel B : anti-RPE65 staining pigmented layer in rat retina.
Panel C : ant-CPLX3 staining ribbon synapses of photoreceptors and bipolar cells in rat retina.
Panel D : ant-ABCA4 staining photoreceptor outer segments in rat retina.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab231782, ab308463 and ab303504 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-RPE65 antibody [EPR22579-44] (AB231782)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse retina tissue staining RPE65 with ab231782 at a 1/8000 dilution, ab140603 anti-CPX used at 1/2000 dilution and ab221664 anti-RHO used at a 1/30000 dilution.

Panel A : merged staining of anti-RPE65 (green; Opal™520), anti-CPX (magenta; Opal™690) and anti-RHO (gray; Opal™570) on mouse retina.

Panel B : anti-RPE65 staining pigmented layer in mouse retina.

Panel C : ant-CPX staining outer nuclear layer and inner nuclear layer in mouse retina.

Panel D : ant-RHO staining rod-shaped photoreceptor cells in mouse retina.

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

The section was incubated in three rounds of staining : in the order of ab231782, ab140603 and ab221664 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

Supplier Data

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) (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 (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

false

• WB

Supplier Data

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 60 kDa

Observed band size: 65 kDa

false

• WB

Lab

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] (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

Observed band size: 65 kDa

false