Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Granzyme B with ab255598 at 1/3000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes in human tonsil (PMID: 29042640) is observed.

The section was incubated with ab255598 for 30 mins at RT.

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

Counterstained with Hematoxylin.

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

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).

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Granzyme B with ab255598 at 1/3000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes in human colon is observed.

The section was incubated with ab255598 for 30 mins at RT.

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

Counterstained with Hematoxylin.

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

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).

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Mouse spleen tissue labeling ab255598 with Granzyme B at 1/200 dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution. The nuclear counterstain was DAPI (Blue).

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

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

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat spleen tissue labeling ab255598 with Granzyme B at 1/200 dilution followed by ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary at 1/1000 dilution. The nuclear counterstain was DAPI (Blue).

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor 488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Granzyme B with ab255598 at 1/3000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes in mouse lung (PMID: 23642129) is observed.

The section was incubated with ab255598 for 30 mins at RT.

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

Counterstained with Hematoxylin.

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

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).

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Granzyme B with ab255598 at 1/3000 dilution  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes in mouse spleen (PMID: 7697916) is observed.

The section was incubated with ab255598 for 30 mins at RT.

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

Counterstained with Hematoxylin.

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

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).

Granzyme B was immunoprecipitated from 0.35 mg mouse spleen cells (treated with 2.5ug/ml Concanavalin A for 72h) whole cell lysate with ab255598 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab255598 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: Mouse spleen cells (treated with 2.5ug/ml Concanavalin A for 72h) whole cell lysate 10μg

Lane 2: ab255598 IP in mouse spleen cells (treated with 2.5ug/ml Concanavalin A for 72h) whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255598 in mouse spleen cells (treated with 2.5ug/ml Concanavalin A for 72h) whole cell lysate.

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

Exposure time: 10 seconds

All lanes : Anti-Granzyme B antibody [EPR22645-206] (ab255598) at 1/1000 dilution

Lane 1 : Untreated mouse splenocyte lysate
Lane 2 : Mouse splenocytes treated with 2.5µg/ml concanavalin for 72 hours, lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 27 kDa
Observed band size: 28,65 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

The expression of Granzyme B is elevated in stimulated splenocytes. Granzyme B forms complex with its protease inhibitor.

The molecular weight observed is consistent with what has been described in the literature (PMID: 8663264, 17116752, 21919028, 9774654).

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

Anti-Granzyme B antibody [EPR22645-206] (ab255598) at 1/1000 dilution + NK-92 (human peripheral blood malignant non-Hodgkin's lymphoma cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 1 second

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