Anti-CNPase antibody [11-5B] (ab6319)

ab6319 staining CNPase in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab6319 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

Lanes 1 - 3: Merged signal (red and green). Green - ab6319 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab6319 was shown to recognize CNPase in wild-type HAP1 cells as signal was lost at the expected MW in CNPase knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CNPase knockout samples were subjected to SDS-PAGE. Ab6319 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

IHC-P image of CNPase staining on rat brain sections using ab6319 (1/1600). heat mediated antigen retrieval on paraffin embedded sections was performed using citric acid. The sections were then blocked with 1% BSA for 10 min at 21°C. The primary antibody was incubated for 16 hours at 21°C. The sections were then incubated in Goat anti-mouse (Biotin) at 1:200.

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Lanes 1- 2: Merged signal (red and green). Green - ab6319 observed at 48 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

 ab6319 was shown to react with CNPase in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264949 (knockout cell lysate ab256877) was used. Wild-type HeLa and CNP knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab6319 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 5 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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Overlay histogram showing SH-SY5Y cells stained with ab6319 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab6319, 2µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

IHC image of CNPase staining in human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. 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 ab6319, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer 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.

ab6319 staining CNPase in the rat oligodendrocytes by ICC/IF (Immunoytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with methanol and blocked with 5% BSA for 1 hour at 37°C. Samples were incubated with primary antibody (1/100 in PBS) for 18 hours at 4°C. An Alexa Fluor^® 594-conjugated Goat anti-mouse IgG polyclonal (1:200) was used as the secondary antibody.

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ab6319 stained in SKNSH cells. Cells were fixed with 100% methanol (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6319 at 10µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

ab6319 staining CNPase in Dog Cerebellum tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1500 in blocking buffer) for 2 hours at 21°C. A Biotin-conjugated Goat anti-mouset IgG polyclonal (1/200) was used as the secondary antibody.

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