Anti-PGP9.5 antibody [13C4 / I3C4] (ab8189)

ab8189 staining PGP9.5 in primary rat neurons/glia, DIV14 (prepared from E18 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDHEP) cells. The cells were fixed with 4% paraformaldehyde (10 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 ab8189 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150121, Goat polyclonal Secondary Antibody to Mouse IgM - mu chain (Alexa Fluor^® 488) 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).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

IHC-P image of PGP9.5 staining on P5 mouse skin sections using ab8189 (1/1000).

The sections were de-paraffinized and subjected to heat meadiated antigen retrieval using citric acid. The sections were then blocked using 1% BSA for 10 mins at 21°C. ab8189 was then incubated for 16 hours at 21°C. The secondary antibody used was Got polyclonal to anti-mouse IgG conjugated to biotin (1/200).

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Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: PGP9.5 knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab8189 observed at 25 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab8189 was shown to specifically react with PGP9.5 in wild-type HAP1 cells as signal was lost in PGP9.5 knockout cells. Wild-type and PGP9.5 knockout samples were subjected to SDS-PAGE. ab8189 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 PGP9.5 staining on zebrafish brain using ab8189 (1/1000). The sections were subjected to heat mediated antigen retrieval using citric acid. The sections were then blocked using 1% BSA for 10 mins for 21°C. The primary antibody (ab8189) was incubated at a dilution of 1/1000 at 21°C for 16 hours. The secondary antibody used was undiluted goat polyclonal to Mouse IgG conjugated to biotin.

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IHC image of PGP9.5 staining in rat pancreas formalin-fixed, paraffin-embedded tissue section, performed on a Leica Bond^TM 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 ab8189, 0.02µg/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 hematoxylin 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.