Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues

Abstract

Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed paraffin-embedded (FFPE) tissue have been less successful. Given that FFPE remains the most common method to fix and analyze mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective antigen retrieval buffer. We also validated 22 antibodies specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique antibodies are currently not available. Lastly, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and antibody panels in the quantitation and spatial distribution of immune cells.

Targeting TGF βR2‐mutant tumors exposes vulnerabilities to stromal TGF β blockade in pancreatic cancer

“…Formalin‐fixed, paraffin‐embedded tissues were cut in 5‐μm sections. Sections were evaluated by immunohistochemical analysis following our previously reported protocol (Sorrelle et al , ) using antibodies specific for anti‐P‐STAT3 (Tyr705, #9145, Cell Signaling, dilution 1:500), anti‐ARG1 (#93668, Cell Signaling, dilution 1:500), anti‐CD8α (#98941, Cell Signaling, dilution 1:1,000), anti‐ZAP70 (#2705, Cell Signaling, dilution 1:500), anti‐P‐SMAD2 (Ser465/467, AB3849, Millipore, dilution 1:500), anti‐IL‐6Rα (AF1830, R&D Systems, dilution 1:250), anti‐iNOS (PA1‐21054, Thermo Fisher Scientific, dilution 1:200), anti‐CD3 (PA1‐29547, Thermo Fisher Scientific, dilution 1:1,000), anti‐F4/80 (NBP2‐12506, Novus Biologicals, 1:200), anti‐FOXP3 (MAB8214, Novus Biologicals, dilution 1:500), anti‐CD11b (ab133357, Abcam, dilution 1:1,000), and anti‐Ki67 (ab15580, Abcam, dilution 1:1,000). Images were obtained with a Nikon Eclipse E600 microscope using a Niko Digital Dx1200me camera and ACT1 software (Universal Imaging Corporation).…”

“…Formalin-fixed, paraffin-embedded tissues were cut in 5-lm sections. Sections were evaluated by immunohistochemical analysis following our previously reported protocol (Sorrelle et al, 2019) using antibodies specific for anti-P-STAT3 (Tyr705, #9145, Cell Signaling, dilution 1:500), anti-ARG1 (#93668, Cell Signaling, dilution 1:500), anti-CD8a (#98941, Cell Signaling, dilution 1:1,000), anti-ZAP70 (#2705, Cell Signaling, dilution 1:500), anti-P-SMAD2 (Ser465/467, AB3849, Millipore, dilution 1:500), anti-IL-6Ra (AF1830, R&D Systems, dilution 1:250), anti-iNOS (PA1-21054, Thermo Fisher Scientific, dilution 1:200), anti-CD3 (PA1-29547, Thermo Fisher Scientific, dilution 1:1,000), anti-F4/80 (NBP2-12506, The paper explained Problem TGFb is a druggable target, inhibition of which has shown efficacy in multiple tumor types especially in combination with immune checkpoint blockade. However, inhibition of the TGFb signaling pathway in pancreatic cancer (PDA) has not been successful in preclinical or clinical studies.…”

Multiplex immunofluorescence methods in neurodegenerative diseases

“…Although stripping techniques may either elute or denaturate antibodies, both terms are used interchangeably in the literature. These seven strategies are: (1) heatbased, particularly common in multiplex protocols using TSA (Ilie et al, 2018;Jufas et al, 2008;Lan et al, 1995;Lim et al, 2018;Mansfield, 2017;Parra et al, 2017;Roy et al, 2019;Saylor et al, 2018;Sorrelle et al, 2019;Stack et al, 2014;Toth and Mezey, 2007;Wegner et al, 2017;Zhang et al, 2017);…”

“…(2) a glycine/SDS solution at a low pH (Bolognesi et al, 2017;Gut et al, 2018;Nakane, 1968;Narhi et al, 1997a;Pirici et al, 2009;Sorrelle et al, 2019) 2014) which reduces the disulfide bonds present in antibodies, thus breaking down their tertiary structure (Capel et al, 1980;Crivianu-Gaita et al, 2015); (5) a low-pH oxidizing solution of KMnO 4 and H 2 SO 4 (Glass et al, 2009;Tramu et al, 1978); (6) chaotropic salts (Bolognesi et al, 2017;Gut et al, 2018;Narhi et al, 1997a;Narhi et al, 1997b); (7) combining antibodies with drastically different abundances (Wang et al, 1999).…”

“…The advent of tyramide signal amplification (TSA) improved the options for combining conspecific antibodies in the same sample while providing signal amplification if combined with an antibody stripping technique. (Buchwalow et al, 2018;Chao et al, 1996;Dixon et al, 2015;Lim et al, 2018;Mansfield, 2017;Pirici et al, 2009;Roy et al, 2019;Sorrelle et al, 2019;Stack et al, 2014;Wang et al, 1999;Zhang et al, 2017). In short, TSA uses a peroxidase-mediated reaction to covalently deposit fluorophores or chromogens to tyrosine side chains proximal to the target epitope (Lim et al, 2018;Wang et al, 1999).…”