Anti-Sortilin/NT3 antibody [EPR23093-58]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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.

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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

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

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (ab263864) at 1/1000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse brain lysate at 20 µg

Lane 4:

Mouse lung lysate at 20 µg

Lane 5:

Rat brain lysate at 20 µg

Lane 6:

Rat lung lysate at 20 µg

Lane 7:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lanes 2 - 7:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 92 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

Western blot : Anti-Sortilin/NT3 antibody [EPR23093-58] ab263864 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 100 kDa in Wild-type HeLa cell lysates with no signal observed at this size in SORT1 knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% Milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (ab263864) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysates at 20 µg

Lane 2:

Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell line (<a href='/ko/products/cell-lines/human-sort1-sortilin-nt3-knockout-hela-cell-line-ab264772'>ab264772</a>) at 20 µg

Lane 3:

SW480 cell lysates at 20 µg

Lane 4:

SH-SY5Y cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 100 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (AB263864)

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

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR23093-58] (ab263864) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 92 kDa

Observed band size: 100 kDa

false