Dako envision system hrp labeled polymer anti rabbit

Tissues were fixed in 10% formalin and paraffin-embedded. Slides were sectioned at 4 µm interval and all subsequent steps were carried out at RT. Heat-induced epitope retrieval was carried out using a Menarini Access Retrieval Unit with a sodium citrate buffer (pH 6) for 1 min 40 s at 125°C, full pressure. The slides were then loaded onto a Dako Autostainer and rinsed with a Tris/Tween buffer (pH 7.5) before treatment with Dako Real TM Peroxidase blocking solution (Agilent Technologies, cat. no. S202386-2) for 5 min followed by buffer rinse (Tris/Tween, pH 7.5) for an additional 5 min. Slides were then treated with the primary antibody: Polyclonal Rabbit Anti-Human CD3 (Agilent Technologies, cat. no. A045201-2) diluted in Dako universal diluent (Agilent Technologies, cat. no. S080981-2) and stained for 30 min. Two rounds of 5 min buffer rinse (Tris/Tween, pH 7.5) were carried out before secondary staining with Dako EnVision + System-HRP Labeled Polymer Anti rabbit (Agilent Technologies, cat. no. K400211-2) for 30 min. The slides were then rinsed twice (Tris/Tween, pH 7.5) and treated with 3,3′-diaminobenzidine + substrate-chromogen system (Agilent Technologies, cat. no. K346889-2) for 10 min. Finally, the slides were washed thrice in H₂O and counterstained with Gills Hematoxylin (Sigma-Aldrich, cat. no. GHS1128) for 27 s followed by additional wash in H₂O.

Five-μm-thick ice sections were stained with hematoxylin and eosin staining or subjected to immunohistology with an anti-α smooth muscle actin (α-SMA) antibody (ab5694, Abcam Japan, Tokyo, Japan), an anti-neutrophil antibody (ab2557, Abcam Japan, Tokyo, Japan), and an anti-Mac-3 antibody (550292, BD Pharmingen, Tokyo, Japan). To detect primary antibodies, sections for the anti-α-SMA antibody were incubated with the Dako Envision+ system HRP-labeled polymer anti-rabbit (ready to use) (Dako North America, California, USA) and sections for the anti-neutrophil and anti-Mac-3 antibodies were incubated with polyclonal rabbit anti-rat immunoglobulins/HRP (Dako North America, California, USA). These immunohistological staining was performed in accordance with our previous studies [21 (link),25 (link),30 (link),31 (link)]. Negative control slides were obtained by omitting each primary antibody.

Tissue microarrays were constructed by the UCLA Translational Pathology Core Laboratory using a Veridiam VTA-110CC Semiautomated Tissue Arrayer using standard protocols. Tissue microarrays were stained against ALDH1A3 using a rabbit anti-human ALDH1A3 antibody (Thermo Fisher Scientific, PA5-29188) at the dilution of 1:1000 overnight at 4 °C, and against PSMD1 antibody (Abcam, ab2941) at the dilution of 1:500 overnight at 4 °C. The slides were rinsed with PBST and were incubated with Dako EnVision+ System- HRP Labeled Polymer Anti-Rabbit (Dako, K4003) at room temperature for 30 min. After a rinse with PBST, the slides were incubated with DAB (3,3′-diaminobenzidine) for visualization. Subsequently, the slides were washed in tap water, counterstained with Harris’ Hematoxylin, dehydrated in ethanol, and mounted with media. Tumor cores were scored by a board-certified breast cancer pathologist based on ALDH1A3 expression on membranes and cytoplasm or cytoplasmic PSMD1 expression on a scale from 0 to + 3 (0: no reaction, + 1 reaction barely visible, + 2 reaction clearly visible, + 3 reaction intense). ALDH1A1 expression was scored by the same principle.

Primary antibody sources and dilutions (2% BSA) obtained from Abcam included: anti-CD4 (ab183685, 1/200), anti-CRT (ab2907, 1/50), anti-HMGB-1 (ab18256, 1/200), anti-LRP1(CD91) (ab92544, 1/50), anti-TLR4 (ab13867, 1/50), and anti-perforin (ab16074, 1/100). Anti-CD8 (#14-0808, 1/100), anti-Foxp3 (#13-5773, 1/200) and anti-IL-10 (#14-7101, 1/50) were from eBioscience. Anti-cleaved caspase-3 antibody was from Cell Signaling (#9664, 1/200) and anti-IFN-gamma from Novus Biologicals (NBP1-19761, 1/200). Anti-IL-12p70 was purchased from Novus Biologics (NBP1-85564, 1/100), and anti-IDO was from Biolegend (#122402, 1/100) Secondary antibodies included MACH2 Rabbit HRP-Polymer (Biocare Medical, RHRP520L) for IL-10 and TLR4; MACH2 Rabbit AP-Polymer (Biocare Medical, RALP525) for CD91; Dako EnVision + System HRP-labeled polymer Anti-Rabbit (Dako, K4003) for the remaining biomarkers.