All lanes : Anti-PSGL-1 antibody [EPR22504-36] (ab227836) at 1/1000 dilution

Lane 1 : Human lymph node tissue lysate
Lane 2 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 43 kDa

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

Exposure time:

Lane 1: 3 minutes

Lane 2: 81 seconds

The expression profile observed is consistent with what has been described in the literature (PMID: 9660879; PMID: 28515724).

PSGL-1(P-selectin is the P-selectin glycoprotein ligand-1) is a 125KD glycoprtein and the 250KD band represents the dimer.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PSGL-1 with ab227836 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on human tonsil (PMID: 19594630, 16567389). The section was incubated with ab227836 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

 

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling PSGL-1 with ab227836 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on infiltrated leukocytes of human colon (PMID: 19594630). The section was incubated with ab227836 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

 

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling PSGL-1 with ab227836 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on microglial cells of human cerebrum is observed. The section was incubated with ab227836 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (human T cell leukemia T lymphocyte) cells stained for PSGL-1 using ab227836 (Green) at 1/50 dilution, followed by an AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing membranous staining in Jurkat cells. The nuclear counterstain was DAPI (Blue). Tubulin was stained using the Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is an AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cell line labeling PSGL-1 using ab227836 at 1/500 dilution (Right). Cells were stained with rabbit monoclonal IgG (ab172730) (Left) or ab227836 (Right). Then stained with anti-CD3 conjugated to Alexa Fluor^® 647. Gated on viable cells. The secondary antibody was a Goat anti rabbit IgG (Alexa Fluor^® 488, ab150097) at 1/2000 dilution.

PSGL-1 was immunoprecipitated form 0.35 mg of Jurkat (human T cell leukemia T lymphocyte) whole cell lysate using ab227836 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab227836 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary at a 1/1000 dilution.

Lane 1: Jurkat whole cell lysate µg (input).
Lane 2: ab227836 IP in jurkat whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227836 in Jurkat whole cell lysate.

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

Exposure time: 5 seconds.

The expression profile observed is consistent with what has been described in the literature (PMID: 9660879; PMID: 28515724). PSGL-1(P-selectin is the P-selectin glycoprotein ligand-1) is a 125KD glycoprtein and the 250KD band represents the dimer.