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.

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, PH9.0, 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.

Immunofluorescent analysis of 100% methanol-fixed THP-1 (human monocytic leukemia cell line) cells labeling CD68 with ab213363 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on THP-1 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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

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.

Lanes 1-3 & 5 : Anti-CD68 antibody [EPR20545] (ab213363) at 1/1000 dilution
Lane 4 : Anti-CD68 antibody [EPR20545] (ab213363) at 1/5000 dilution

Lane 1 : Human fetal liver lysate at 20 µg
Lane 2 : Human tonsil lysate at 20 µg
Lane 3 : Human fetal spleen lysate at 20 µg
Lane 4 : THP-1 (human monocytic leukemia cell line) whole cell lysate at 10 µg
Lane 5 : U937 (human histiocytic lymphoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 37 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2/4/5: 30 seconds; Lane 3: 3 minutes.

The observed molecular weight is consistent with the literature (PMID:18405323; PMID:11739566; PMID: 16710801).

Immunofluorescent analysis of 100% methanol-fixed U937 (human histiocytic lymphoma cell line) cells labeling CD68 with ab213363 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on U937 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma 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 cervical carcinoma is observed (PMID: 12118106). 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.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD68 with ab213363 at 1/8000 dilution, followed by Goat anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on Kupffer cells of human liver is observed (PMID: 12118106). 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.