All lanes : Anti-Sortilin/NT3 antibody [EPR23093-58] (ab263864) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 2 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: 48 seconds.

All lanes : Anti-Sortilin/NT3 antibody [EPR23093-58] (ab263864) at 1/1000 dilution

Lane 1 : Human brain lysate
Lane 2 : SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate
Lane 3 : Mouse brain lysate
Lane 4 : Mouse lung lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat lung lysate
Lane 7 : Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: Lanes 1-2: 48 seconds; Lanes 3-7: 6 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labeling Sortilin/NT3 with ab263864 at 1/600 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1\2000 was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Sortilin/NT3 with ab263864 at 1/600 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1\2000 was used as the secondary antibody.

Immunofluorescent analysis of 100% methanol-fixed Neuro-2a (Mouse neuroblastoma neuroblast) cells labeling Sortilin/NT3 with ab263864 at 1\100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1\1000 dilution (Green). Confocal image showing golgi staining in Neuro-2a cell line. 100% methanol fixation is recommended. Counterstained with ab169276 Anti-GM130 antibody-Golgi Marker (1/500 dilution) and ab150120 AlexaFluor^®594 Goat anti-Mouse secondary (1/1000 dilution, red). The nuclear counterstain was DAPI (Blue).

The negative controls are as follows:

-ve control 1: ab263864 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution.

-ve control 2: ab169276 Anti-GM130 antibody- Golgi Marker at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution.

Immunofluorescent analysis of 100% methanol-fixed SH-SY5Y (Human neuroblastoma epithelial cell) cells labelling Sortilin/NT3 with ab263864 at 1\100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1\1000 dilution (Green). Confocal image showing cytoplasmic staining in SH-SY5Y cell line. 100% methanol fixation is recommended. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1\1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling Sortilin/NT3 with ab263864 at 1/5000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat cerebrum is observed. The section was incubated with ab263864 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Sortilin/NT3 with ab263864 at 1/5000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse cerebrum iso bserved. The section was incubated with ab263864 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma and the paired adjacent breast tissue tissue labeling Sortilin/NT3 with ab263864 at 1/5000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human breast carcinoma (panel A) and weak staining on the paired adjacent breast tissue (panel B) (PMID: 25871389) is observed. The section was incubated with ab263864 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue labeling Sortilin/NT3 with ab263864 at 1/5000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human ovarian carcinoma (PMID: 23407681) is observed. The section was incubated with ab263864 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling Sortilin/NT3 with ab263864 at 1/5000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human endometrium (PMID: 25662808) is observed. The section was incubated with ab263864 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.