Betazoid dab chromogen

Immunohistochemical analysis was performed using anti-MLH1 (PM220, Biocare Medical, Concord, CA), anti-MSH2 (PM219, Biocare Medical), anti-MSH6 (PM265, Biocare Medical), and anti-PMS2 (PM344, Biocare Medical). Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm sections. Antigen retrieval was performed using 10mM citrate buffer (anti-MSH2) or Tris-EDTA buffer pH 9.0 (anti-MLH1, anti-MSH6, anti-PMS2) heated to boiling for 32 minutes and blocked for endogenous peroxidase with Peroxidazed 1 (PX968, Biocare Medical). Slides were blocked using Background Sniper (BS966, Biocare Medical), then incubated for one hour at room temperature with antibodies diluted 1:100–1:200. Slides were treated with MACH 4 Universal HRP Polymer (M4U534, Biocare Medical), detected with Betazoid DAB Chromogen (BDB2004, Biocare Medical), counterstained with hematoxylin, dehydrated, and cover-slipped. Samples were graded for the absence or presence of nuclear staining of the MMR proteins. Intact staining in the infiltrating immune cells was used as an internal control. A board-certified pathologist who was blinded to the MSI results confirmed all the IHC results.

Immunohistochemistry (IHC) was performed on formalin fixed, paraffin embedded left colon biopsy tissue from healthy and ulcerative colitis patients. The sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 degrees for 4 mins in a decloaking chamber. Endogenous peroxidases were quenched by a 5 min incubation of 10% H₂O₂ in methanol. Non-specific binding was blocked by a 20 min incubation in Biocare background Sniper solution (BS966G, Biocare, Concord, CA21520, USA). Staining for human SLPI was performed using a specific antibody against human SLPI (PA5-20385, rabbit polyclonal, Thermo Fisher Scientific, Rockford, IL 61105, USA) at room temperature for 1 hour. Samples were then incubated with a horseradish peroxidase (HRP)-polymer (MRH53BL10, Biocare, Concord, CA21520, USA) for 30 mins, stained with Betazoid DAB chromogen (BDB900B, Biocare, Concord, CA21520, USA), counterstained with hematoxylin, dehydrated and mount in DPX.

Cecum and liver samples embedded in paraffin were dewaxed at 55° C for 30 min, rehydrated twice in xylol and ethanol at different concentrations (100%, 96%, 80% and 70%), and washed in distilled water. Endogenous peroxidase activity was inhibited by placing the slides in hydrogen peroxide and methanol (3ml/100ml) for 20 min at room temperature (RT). For antigen retrieval, slides were included in heat retrieval solution Diva Decloaker 20X (BioCare Medical), and placed in a pressure cooker for 3 min, tempered for 5 min, and washed with PBS-TWEEN 0.1%. Following the blocking, the slides were incubated with Background Sniper (BioCare Medical) for 15 min at RT and washed with PBS-TWEEN 0.1%. Then, a primary polyclonal anti-Salmonella Typhimurium antibody (BIOSS) diluted 1:300 was applied to the slides, incubated for 2 h at RT, and washed in PBS-TWEEN 0.1%. Subsequently, the slides were exposed to HRP-Polymer (Biocare Medical, USA) for 30 min, washed with PBS-TWEEN 0.1% and, finally, diaminobenzidine (Betazoid DAB Chromogen- Biocare Medical, USA) was applied for 1 min to detect the primary antibody.

Immunohistochemistry was performed on 10% (v/v) formalin fixed, paraffin embedded left colon biopsies. Sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 °C for 4 min in a decloaking chamber. Endogenous peroxidases were quenched by a 5-min incubation in 10% H₂O₂ in methanol. Non-specific binding was blocked by a 20-min incubation in BioCare background Sniper solution (BS966G, BioCare, Concord, CA21520, USA). Immunostaining was performed using specific antibodies against IL-1β (ab9722, Abcam, Cambridge, MA, USA) and NLRP3 (ab17267, Abcam, Cambridge, MA, USA) at room temperature for 1 h. An additional incubation with MACH 1 mouse probe (UP537L10, BioCare, Concord, CA21520, USA) for 15 min at room temperature was performed on sections incubated with the anti-NLRP3 antibody. Samples were then incubated with a horseradish peroxidase (HRP)-polymer (MRH53BL10, BioCare) for 30 min, stained with Betazoid DAB chromogen (BDB900B, BioCare, Concord, CA21520, USA), counterstained with hematoxylin, dehydrated and mounted with distyrene, plasticizer and xylene. Slides were examined using an IX71 microscope (Olympus Australia, Melbourne, Australia).

Immunohistochemistry (IHC) was performed as described previously 12 (link),23 (link),25 (link). Briefly, 5-μm sections were incubated overnight at 4°C with primary antibody diluted in Background Sniper (#BS966M, Biocare Medical, Concorde, CA, USA). Primary antibodies: ABCG2 (BXP-21) (#SC-58222, Santa Cruz Biotechnology Inc., Dallas, TX) at 1:500 dilution, Bmi-1 (1.T.21) (#ab14389, Abcam plc., Cambridge, UK) at 1:100 dilution. Staining utilized MACH 1 Mouse Probe, MACH 1 Universal HRP-Polymer (#M1U539L10; Biocare Medical) with Betazoid DAB chromogen (#BDB2004L; Biocare Medical), CAT haematoxylin (#CATHE-M; Biocare Medical), and Leica CV Mount (Leica, Nussloch, Germany). Positive and negative staining controls for each antibody were conducted (Figure S1).

After tissue slides were prepared, they were incubated with the first antibody goat anti-rabbit HRP polymer (Mach 2 Rabbit HRP-Polymer, Biocare Medical), and Betazoid DAB Chromogen (Biocare Medical) was then used to stain the first marker. The slides were rinsed thoroughly with distilled water, treated with denaturing solution (Biocare Medical) for 3 min to denature anti-rabbit-HRP, and then rinsed for staining with the second antibody. Goat anti-mouse HRP polymer (Mach 2 Mouse HRP-Polymer, Biocare Medical) and Vina Green Chromogen (Biocare Medical) were used for the staining. The slides were finally counterstained with hematoxylin (Biocare Medical).

Deparaffinated and hydrated tissue was blocked for 5 min with 5% hydrogen peroxide, heated for antigen retrieval with Diva Decloaker (Biocare, Tijuana, Mexico) and blocked with Rodent Block R (Biocare, Mexico). Then, the sections were incubated with anti-CD3 (1:1000) (Biocare, Mexico) for 30 min at RT, and revealed with Betazoid DAB Chromogen and Betazoid DAB substrate buffer (Biocare, Mexico). Tissue sections were then incubated in haematoxylin, dehydrated and cover-slipped for microscopic examination.