Anti-GPVI antibody [EPR25690-71]

• ICC

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Immunocytochemistry - Anti-GPVI antibody [EPR25690-71] (AB302950)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEL (human erythroleukemia erythroblast) cells labelling GPVI with ab302950 at 1/100 (4.53 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing increased membranal and cytoplasmic staining in HEL cells treated with Phorbol-12-myristate-13-acetate (10 nM) for 48 hoursImage was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/mL dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC

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Immunohistochemistry - Anti-GPVI antibody [EPR25690-71] (AB302950)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling GPVI with ab302950 at 1/100 (4.53 ug/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on megakaryocytes of mouse spleen. The section was incubated with ab302950 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC

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Immunohistochemistry - Anti-GPVI antibody [EPR25690-71] (AB302950)

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling GPVI with ab302950 at 1/100 (4.53 ug/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Negative control : no staining on mouse pancreas. The section was incubated with ab302950 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-GPVI antibody [EPR25690-71] (AB302950)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse pancreas (fresh) tissue labeling GPVI with ab302950 at 1/500 (9.06 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Negative control : confocal image showing positive staining on mouse pancreas (PMID : 10961879). The nuclear counterstain was DAPI (Blue). The section was incubated with ab302950 for 60mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-GPVI antibody [EPR25690-71] (AB302950)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh) tissue labeling GPVI with ab302950 at 1/500 (9.06 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining on megakaryocytes of mouse spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab302950 for 60mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.<\p> .

• WB

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Western blot - Anti-GPVI antibody [EPR25690-71] (AB302950)

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

Negative control : human pancreas (human protein atlas database).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

The identity of the lower MW band at approximately 45 kDa is unknown.

Exposure time :

Lane 1-3 : 10 seconds

Lane 4-5 : 48 seconds

All lanes:

Western blot - Anti-GPVI antibody [EPR25690-71] (ab302950) at 1/1000 dilution

Lane 1:

Human plasma at 40 µg

Lane 2:

Human testis tissue lysate at 40 µg

Lane 3:

Human pancreas tissue lysate at 40 µg

Lane 4:

Mouse testis tissue lysate at 40 µg

Lane 5:

Rat testis tissue lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Predicted band size: 37 kDa

Observed band size: 60 kDa

true

• WB

Lab

Western blot - Anti-GPVI antibody [EPR25690-71] (AB302950)

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

GPVI is expressed mainly on platelets (PMID : 30601137, PMID : 34496636).

All lanes:

Western blot - Anti-GPVI antibody [EPR25690-71] (ab302950) at 1/1000 dilution

Lane 1:

Mouse platelet tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Mouse bone marrow tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 54 kDa

false

Exposure time: 180s