Anti-PD1 抗体 [CAL20] - BSA and Azide free

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

This data was developed using ab237728, the same antibody clone in a different buffer formulation.

The BOND™ Polymer Refine Detection System on the BOND RX Automated Stainer (DS9800, Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical labelling of formalin-fixed, paraffin-embedded (FFPE) tissues. The protocol includes a peroxide block, post-primary reagent, polymer detection, DAB chromogen, and haematoxylin counterstain, all automated to minimize variability. Antigen retrieval was performed by heat-induced epitope retrieval (HIER) for 20 minutes in Tris-EDTA buffer (BOND Epitope Retrieval Solution 2, AR9640, Leica Biosystems, Wetzlar, Germany). For mouse and rabbit antibodies, the sequence comprised peroxide block (10 min), primary antibody incubation (30 min), post-primary (10 min), polymer (10 min), DAB (10 min), and haematoxylin (8 min). For rat antibodies, an anti-rat step (1 : 300 dilution, Vector Laboratories, California, USA; 20 min) was included prior to polymer application (15 min).

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human tonsil tissue staining PD-L1 with ab205921 at a 1 : 500 (2.19 ug/ml) dilution; PD1 with ab237728 at 1 : 2000 (0.525 ug/ml) dilution and CD68 with ab213363 at 1 : 500 (1.26 ug/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.

Panel A : merged staining of anti-CD68 (magenta; Opal^™690), anti-PD-L1 (green; Opal^™520) and anti-PD1 (red; Opal^™570) on human tonsil.
Panel B : anti-PD-L1 stained on cells involved in T cell inhibition.
Panel C : anti-PD1 stained on antigen-stimulated T cells.
Panel D : anti-CD68 stained on macrophages.

The section was incubated in three rounds of staining : in the order of ab213363 and ab205921 for 30 mins, then ab237728 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Tissue Microarrays stained for "Anti-PD1 antibody [CAL20]" using "ab237728"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 30 minutes. The sections were incubated with ab237728 for 15 mins at room temperature followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Immunohistochemical analysis of paraffin-embeded human tonsil tissue labeling PD1 with ab237728 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the human tonsil.is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins.

The section was incubated with ab237728 for 15 mins at room temperature.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Immunohistochemical analysis of paraffin-embeded Rhesus monkey tonsil tissue labeling PD1 with ab251613 followed by Polink 1 Polymer HRP anti-Rabbit IgG.

Heat mediated antigen retrieval-Buffer/Enzyme Used : Dako pH9.

This image is courtesy of Dr. Chi Ngai Chan

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Formalin-fixed, paraffin-embedded human lung carcinoma tissue stained for PD1 using ab237728 at 0.125 μg/ml dilution in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MOLT-4 (human lymphoblastic leukemia cell line) cells labeling PD1 with ab237728 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Flu^or® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in MOLT-4 cells treated with PMA (10ng/ml 24h) and Ionomycin(500ng/ml 24h). The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Flu^or® 488) (ab150077) secondary antibody at 1/1000 dilution.

Cells were either untreated, ot treated with treated with PMA (10ng/ml 24h) and Ionomycin (500ng/ml 24h).

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] - BSA and Azide free (AB251613)

Formalin-fixed, paraffin-embedded human lymph node tissue stained for PD1 using ab237728 at 0.125 μg/ml dilution in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237728).