Universal hier antigen retrieval reagent

Sequential immunostaining was performed on 3-μm-thick Formalin-Fixed Paraffin-Embedded (FFPE) murine left lung sections as previously described⁸⁰ . Briefly, heat mediated antigen retrieval was performed in Universal HIER antigen retrieval reagent (Abcam, #ab208572). Antibody elution was performed between each staining cycle. Primary antibodies used were: rabbit anti-5-Lipoxygenase/5-LO (1:500, Abcam #ab169755), rabbit anti-Myeloperoxidase (1:1000, Abcam #ab208670), rabbit anti-IBA1 (1:1000, VWR #100369–764), rabbit anti-iNOS (1:250, Abcam #ab239990), and rabbit anti-CD206 (1:250, Abcam #ab64693). Secondary antibodies used were: anti-rabbit 555 (1:500, Cell Signaling #4413 S) and anti-rabbit 647 (1:1000, Cell Signaling #4414 S).
Acquired images were processed using FIJI and the FIJI plugin HyperStackReg V5.6^{81 (link)} (and Ved Sharma. 2018, December 13 Zenodo. 10.5281/zenodo.2252521). Autofluorescence acquired in nonrelevant channels was substracted as appropriate. Quantitation was performed using Ilastik (v1.3.3post3)^{82 (link)} and CellProfiler (4.1.3)^{83 (link)}.

Immunohistochemistry was performed as previously described [22 (link)]. For immunostaining, mouse tissues were fixed with Mildform (Wako Pure Chemical, Tokyo, Japan). For human renal tumor tissues, a prefixed human tissue array was purchased (KD2082a, US Biomax Inc., Rockville, MD, USA). After paraffinization, the tissues were sectioned and deparaffinized using xylene and ethanol, followed by antigen retrieval using Universal HIER Antigen Retrieval Reagent (Abcam). Samples were subjected to hematoxylin & eosin staining or immunostaining. After blocking with Block ACE (Bio‐Rad, Hercules, CA, USA), the samples were incubated with primary antibodies and the appropriate secondary antibodies (Table S4) and stained using a Dako Liquid DAB+ Substrate Chromogen System (Agilent Technologies, Santa Clara, CA, USA) and Mayor’s hematoxylin. Images were captured using an AX80 microscope (Olympus, Tokyo, Japan).
To detect apoptosis, In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics) and DAPI Fluoromount‐G (Southern Biotech, Birmingham, AL, USA) were used as previously described [32 (link)]. Fluorescent images were captured using a BZ‐X710 microscope (KEYENCE, Osaka, Japan).

The procedure used for aortic immunofluorescence staining was similar to that described in our previous studies [12 (link),29 (link),30 (link),31 (link)]. Briefly, paraffin thoracic aortic sections were deparaffined with xylene and rehydrated with ethanol, followed by antigen retrieval in a Universal HIER antigen retrieval reagent (Abcam, Cambridge, UK, ab208572). After blocking the sections, they were incubated with the indicated primary antibodies or respective IgG controls diluted in blocking buffer in a cold room (4 °C) overnight. The tissue sections were then washed and subsequently incubated with an appropriate Alexa Fluor Plus 2nd antibody (1:1000 dilution), followed by nuclei staining with 4,6-diamidino-2-phenylindole (DAPI) (1 ug/mL). After mounting, the slides were examined using a laser scanning confocal microscope (Zeiss LSM 510 Mark 4) and Zen 2009 image software. The mean fluorescence intensity (MFI) for target proteins and DAPI of the aortic wall from each section were measured with Image J pro software by two experienced investigators blinded to the treatments, and presented as the relative MFI (target proteins over DAPI). Alternatively, cells stained positive for the target proteins and DAPI (total cells) were counted by two experienced investigators blinded to the treatments. Three sections were analyzed per vessel sample and averaged.

The procedure used for aortic immunofluorescence staining was similar to that described in our previous studies.^{27 (link)–30 (link)} Briefly, paraffin aortic sections were deparaffined with xylene and rehydrated with ethanol, followed by antigen retrieval in a Universal HIER antigen retrieval reagent (ab208572; Abcam). After blocking, the sections were incubated with indicated primary antibodies or respective IgG controls diluted in blocking buffer in a cold room (4 °C) overnight. The tissue sections were then washed and subsequently incubated with an appropriate Alexa Fluor Plus second antibody (1:1000 dilution), followed by nuclei staining with DAPI (4,6-diamidino-2-phenylindole; 1 µg/mL). After mounting, the slides were examined using a laser scanning confocal microscope (Zeiss LSM 510 Mark 4) and Zen 2009 image software. The mean fluorescence intensity for target proteins and DAPI of the aortic wall from each section were measured with the Image J pro software by 2 experienced investigators blinded to the treatments and presented as the relative mean fluorescence intensity (target proteins over DAPI). Alternatively, cells stained positive for the target proteins and DAPI (total cells) were counted by 2 experienced investigators blinded to the treatments. Three sections were analyzed per vessel sample and averaged.

We performed immunohistochemistry (IHC) staining of FFPE tissue sections for PD-L1 protein using an anti-PD-L1 antibody (clone 28-8; Cat#ab205921; Abcam; 1:300). Briefly, slides were incubated at 60 °C, deparaffinized in xylenes, and rehydrated with graded ethanol. Antigen retrieval was performed using the Universal HIER antigen retrieval reagent (Cat#ab208572; Abcam; 1:10) in a steamer. Non-specific binding was blocked with the Dako EnVision FLEX Peroxidase-Blocking Reagent. All other staining was performed primarily with Dako series reagents (Cat#K8002; Dako; Undiluted). All slides were counterstained with hematoxylin. Specimens were scored as positive by the pathologist using the  Tumor Proportion Score (TPS), which is the percentage of viable tumor cells with partial or complete membrane staining at any intensity. PD-L1 positivity in the study was defined as TPS ≥ 1%, and the specimens with 1–50% TPS and ≥50% TPS were respectively scored as weakly and strongly positive, respectively.

Whole tumor Sects. (4-μm-thick) of formalin-fixed paraffin-embedded tissue were deparaffinized in xylene and rehydrated in ethanol and distilled water. Antigen retrieval was performed by microwaving sections in Universal HIER antigen retrieval reagent (Abcam) for 20 min. Endogenous peroxidase activity was blocked by treatment with Peroxidase Block (DAKO, Santa Clara, CA, USA) for 10 min, and non-specific binding was blocked by treatment with Protein Block serum-free Ready-to-use (DAKO) for 10 min at room temperature. The sections were then incubated overnight at 4 °C with anti-human PD-L1 monoclonal antibody (clone 28–8, 1:500; Abcam). The sections were then incubated with Rabbit-specific IHC polymer detection kit and visualized using DAB substrate (both Abcam). Finally, sections were counterstained with hematoxylin. For negative control staining, the anti-PD-L1 primary antibody was replaced with a rabbit IgG monoclonal antibody (Abcam).