Envision detection system

Staining was performed in 4 μm formalin-fixed, paraffin-embedded tissue sections of CRCs and matched normal mucosa from 10 patients (Additional file 1: Table S5) with the use of Envision Detection System (DAKO). Sections were deparaffinized with xylene and rehydrated in a series of decreasing concentration of ethanol solutions. Heat-induced epitope retrieval was carried out in Target Retrieval Solution (pH 6) (DAKO) in a 96°C water bath, for 20 minutes. After cooling retrieval solutions for 25 minutes at room temperature, the slides were treated for 5 minutes with Blocker of Endogenous Peroxidase (DAKO). Slides were incubated with anti-H3K27Ac (ab4729, Abcam) (diluted 1:500) for 30 minutes at room temperature and subsequently labeled with the Envision Detection System (DAKO). Color reaction product was developed with 3,3′-diaminobenzidine, tetrahydrochloride (DAB) (DAKO) as a substrate, and nuclear contrast was achieved with hematoxylin counterstaining. Representative pictures of each sample were taken at magnification x400 and used for the automatic calculation of the percentage of positively stained nuclear area (labeling index). For this purpose ImmunoRatio i.e. softwere for automated image analysis [37 (link)] was used and the results for normal samples and CRC sections were compared.

β-catenin expression in tissues without polyps was analyzed using IHC. A piece of tissue from the internal end of distal colon, approximately 1 cm wide, was collected from rats in groups M7 to M10. Half of each tissue sample was fixed in 4% neutral buffered formalin for 24 h, paraffin embedded and cut into 4 μm thick sections. IHC of colon tissues was performed by using an EnVision^TM Detection System (Dako, Troy, MI, USA), according to manufacturer’s instructions. Briefly, tissue sections were mounted onto silanized charged slides and allowed to dry for 1 h at room temperature, followed by 1 h in a 60 °C incubator. After deparaffinization and rehydration, slides were incubated with a proteinase K solution (20 μg/mL⁻¹) for 5 min. After washing with distilled water, tissue sections were immersed in 3% H₂O₂ for 5 min to block endogenous peroxidase, followed by additional washing with buffer. Slides were incubated with a rabbit anti-β-catenin primary antibody (Abcam, Cambridge, UK) for 30 min in a humid chamber. After 2 rinses in buffer, slides were incubated with the detection system for 30 min. Staining was visualized with a 2,4-diaminobutyric Acid (DAB) substrate chromogen solution. Sections were dehydrated, mounted with cover glass, and observed using light microscopy (DFC425C, Leica, Wetzlar, Germany).

Immunostaining was applied on 5 μm thick paraffin sections. After deparaffinization and rehydration, the sections were treated by microwave for 20 min at 400 W in citrate buffer (pH 6.0). After antigen retrieval, samples were incubated for 1 hour at room temperature with primary antibody for nestin (Santa Cruz, USA, sc-23927, dilution 1:100). Sections were then treated with EnVisionTM Detection System (DAKO, Germany) using 3-amino-9-ethylcarbazole (AEC) as substrate, and counterstained with hematoxylin. Negative controls were performed by omitting the first antibody and stained by the EnVisionTM method, and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. The slides were evaluated using a light microscope BX53 with DP12-CCD camera (Olympus, Germany).

Immunohistochemical staining of the tissue arrays was performed according to standard protocols.[19 (link)] In brief, slides were deparaffinized and, after heat-induced antigen retrieval and blocking, incubated with the primary antibody. The primary antibodies and staining conditions were as follows: TGF-Δ1: Acris, #DM1047, dilution 1:100, pretreatment in EDTA-buffer for 36 min.; TGF-Δ2: Acris, #AP15815PU-S, 1:25, EDTA-buffer for 36 min.; Santa Cruz, #sc-90, 1:25, citrate-buffer for 30 min.; p-Smad2/3: Cell Signaling, #3101, 1:200, citrate-buffer for 20 min. Immunoreactivity was detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a chromogen. Stainings for p-Smad2/3 and TGF-Δ2 (for the Santa Cruz antibody) were performed manually using the EnVision^TM Detection System (Dako, # K406511-2). Stainings for TGF-Δ1 and TGF-Δ2 (Acris antibody) were performed on the Benchmark Ultra Autostainer (Ventana/Roche, Mannheim, Germany) using the reagents and detection systems supplied by the vendor. As positive controls we used human placenta tissue for both TGF-Δ2 antibodies, human tonsil tissue for TGF-Δ1 staining and cirrhotic liver tissue for p-Smad2/3 detection.

Paraffin-embedded synovial tissues of RA were sectioned, deparaffinized, and antigen-retrieved with 0.01 M citric acid of pH 6.0. The clinical background of the patients is shown in Supplementary Table 3. Immunohistochemistry was performed with Sox4 antibody (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:100) and detected with EnVisionTM Detection System (DAKO). ELS formation (Score E) and the presence of Sox4-positive cells within the infiltrating cell population without ELSs (Score SN) or with ELSs (Score SE) were assessed by a semi-quantitative four-point scale (none, 0; mild, 1; moderate, 2; and severe, 3). The total Sox4 expression score corresponds with the sum of the SN and SE scores. Triple immunofluorescence staining was performed with CXCL13 antibody (BCA1, rabbit polyclonal, Thermo Fisher, 1:100), Sox4 (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:30), CD3 (Clone PS1, mouse monoclonal, Leica Microsystems, 1:200), CD4 (Clone 1F6, mouse monoclonal, Leica Microsystems, 1:100), CXCR5 (Clone 51505, mouse monoclonal, R&D Systems, 1:1000), and PD-1 (Clone NAT105, mouse monoclonal, Abcam, 1:50) and detected with TSA Plus Fluorescence Kit (PerkinElmer, Inc.). Fluorescence imaging analysis was performed using the FSX100 Fluorescence Microscope (Olympus).

Tissue sections were incubated at 60°C for 10 min to facilitate tissue adherence onto the slides before deparaffinization in two changes of xylene substitute (Sigma-Aldrich Co., St Louis, MO, USA) each for 15 min. This was followed by serial rehydration in graded ethanol (GmbH, Hamburg, Germany) from 100% ethanol followed by 70%, 50% and 30% ethanol, and finally in distilled water. Heat-mediated antigen retrieval was conducted in Tris-EDTA buffer (pH 9.0) using a microwave pressure cooker for 10 min followed by incubation with a mouse anti-TRPM4 monoclonal antibody (clone 10H5; Abcam, Cambridge, UK) at 1:500 dilution (1.536 μg/ml) for one hour at room temperature. Binding of the anti-TRPM4 antibody was detected using HRP-conjugated secondary anti-mouse/rabbit antibody from EnVision^TM detection system (DakoCytomation, Carpinteria, CA, USA) for 30 min and developed with DAB as the chromogen for 5 min. The sections were counterstained with fresh Gill No. 2 hematoxylin solution (Sigma Aldrich) for 10 sec and mounted with the VectaMount^TM (Vector Labs, Burlingame, California) non-aqueous mounting medium.