Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma cell line) cells labeling Chromogranin A with ab254322 at 1/100 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining in Neuro-2a cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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

Immunohistochemical analysis of paraffin-embedded rat panceas tissue labeling Chromogranin A with ab254322 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat pancreas islet (PMID: 22412953) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

The section was incubated with ab254322 for 30 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

 

All lanes : Anti-Chromogranin A antibody [EPR22537-248] (ab254322) at 1/1000 dilution

Lane 1 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : Mouse serum
Lane 4 : Mouse adrenal gland lysate
Lane 5 : Rat adrenal gland lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 50 kDa
Observed band size: 20,75 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 15 seconds; Lane 2: 3.25 seconds; Lane 3:26 seconds; Lane 4: 3.25 seconds; Lane 5: 15 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 14734658, 21052719).

Flow cytometric analysis of  4% paraformaldehyde-fixed, 90% methanol-permeabilized Neuro-2a (mouse neuroblastoma cell line) cells labeling Chromogranin A with ab254322 at 1/60 (red) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue labeling Chromogranin A with ab254322 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse pancreas islet (PMID: 22412953) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

The section was incubated with ab254322 for 30 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

 

Frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat colon tissue labeling Chromogranin A with ab254322 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/1000 dilution (green). Positive staining on enteroendocrine cells of mouse colon (PMID: 27287799) 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).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell line) cells labeling Chromogranin A with ab254322 at 1/100 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining in PC-12 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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

Frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse colon tissue labeling Chromogranin A with ab254322 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/1000 dilution (green). Positive staining on enteroendocrine cells of mouse colon (PMID: 27287799). 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).