Immunohistochemical analysis of paraffin-embedded human liver tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on Kupffer cells of human liver (PMID: 27192565) 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on both infiltrated immunocytes and tumor cells of human lung cancer (PMID: 22586058) 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin lymphoma tissue labeling CSF-1-R with ab229188 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Cytoplasmic and membranous staining of human non-Hodgkin lymphoma cells is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CSF-1-R with ab229188 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Cytoplasmic staining in macrophages of human tonsil (PMID: 26066800) is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling CSF-1-R with ab229188 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Cytoplasmic staining in macrophages infiltrating human lung cancer is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical staining of paraffin embedded human spleen with purified ab133357 at a 1/4000 dilution. The secondary antibody used is a HRP goat anti-rabbit (ab97051). The sample is counterstained with hematoxylin.

Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

IHC image of CD11b staining in a formalin fixed, paraffin embedded human normal spleen tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab133357 at 1/4000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Formalin-fixed, paraffin-embedded human lung stained for iNOS using ab115819 at a dilution of 1/100 in immunohistochemical analysis.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling iNOS with Purified ab115819 at 1/100 dilution (0.65 µg/ml).

Heat mediated antigen retrieval was performed using sodium citrate buffer, pH 6.0.

Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody.

Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Formalin-fixed, paraffin-embedded human lung stained for iNOS using ab115819 at a dilution of 1/100 in immunohistochemical analysis.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung tissue sections labeling iNOS with Purified ab115819 at 1/100 dilution (0.65 µg/ml).

Heat mediated antigen retrieval was performed using sodium citrate buffer, pH 6.0.

Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody.

Negative control: PBS instead of the primary antibody.

Hematoxylin was used as a counterstain.

ab133543 staining liver arginase in paraffin embedded human hepatocellular cancer tissue sections by Immunohistochemistry.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Samples were incubated with primary antibody at 1:2000 dilution (0.13 μg/ml).

A ready to use Goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody.

Hematoxylin was used as a counterstain.

Cytoplasmic and nuclear staining on human hepatocellular cancer.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Kupffer cells of human liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

Formaldehyde-fixed, non-permeabilized human placenta tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Goat anti Rabbit polyclonal HRP conjugate was used as the secondary at a 1/200 dilution.

Heat mediated antigen retrieval buffer/enzyme used: pH 6.0 citrate buffer.

Blocking step: 3% serum for 30 mins at 20°C.

10% Formalin-fixed, non-permeabilized human breast carcinoma tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Donkey anti Rabbit IgG polyclonal AlexaFluor^®647 conjugate was used as the secondary at a 1/200 dilution (red).

Heat mediated antigen retrieval buffer/enzyme used: Tris/EDTA pH 9.0.

Blocking step: 5% serum for 1 hour at 22°C.

Formaldehyde-fixed, non-permeabilized human tonsil tissue stained for CD163 with ab182422 (30 mins at a 1/400 dilution) in immunohistochemical analysis. A Goat polyclonal HRP conjugate was used as the secondary.

Heat mediated antigen retrieval buffer/enzyme used: pH 9.0 EDTA.

Blocking step: 1% ab64226 for 10 mins at RT.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Mannose Receptor with ab252921 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in macrophages (Kupffer cells) of human liver (PMID: 26938527) is observed. Counterstained 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Mannose Receptor with ab252921 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in macrophages of human colon (PMID: 9060831) is observed. Counterstained 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human Karposi's sarcoma tissue labeling Mannose Receptor with ab252921 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in tumor cells of human Kaposi’s Sarcoma (PMID: 9060831) is observed. Counterstained 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue.

Panel A:  Merged staining of Collagen VI (ab182744; green), anti-CD68 (ab213363; red) and anti-Lamin B1 (ab229025; magenta).

Panel B: Anti-Collagen VI (green) stained on extracellular matrix.

Panel C: Anti-CD68 (red) stained on Kupffer cells.

Panel D: Anti-Lamin B1 (magenta) stained on nuclear envelope.

Key protocol steps:  The section was incubated in three rounds of staining with ab182744 (1/1000 dilution), ab213363 (1/1000 dilution) and ab229025 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by tyramide signal amplification with the appropriate fluorophore. Heat mediated antigen retrieval was used (Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins after every round of antibody/fluorophore staining.

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

DAPI was used as a nuclear counter stain. A ready-to-use anti-Rabbit and Mouse Polymer HRP was used as a secondary.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue, labeling CD68 with ab213363 at 1/8000 dilution, followed by Goat anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on macrophages of human tonsil is observed (PMID: 19543531). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD68 with ab213363 at 1/5000 dilution. No blocking step performed. Anti-Rabbit HRP polymer was used as the secondary detection system. Heat-mediated antigen retrieval was performed using EDTA based pH 9.0 buffer.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human spleen tissue labelling PDI with ab243644 at 1.02 µg/mL (B), PD-L 1 with ab213524 at 1/100 dilution (C) and CD68 with ab213363 at 1/300 dilution (D). Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity. 

Panel A: merged staining of anti- PD1 (green, Opal™520), anti- PD-L1 (red, Opal™570) and anti- CD68 (yellow, Opal™690).

Panel B: Anti- PD1 stained on antigen-stimulated T cells.

Panel C: anti- PD-L1 stained on cells involved in T cell inhibition

Panel D: anti-CD68 stained on macrophages.

The section was incubated in three rounds of staining: in the order of ab243644, ab213363 and ab213524 for 30 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.