3 3 diaminobenzidine (dab)

Sectioned slides were deparaffinized, then proceeded to heat induced antigen retrieval with sodium citrate (pH 6). Endogenous peroxidase activity was then quenched with 3% H₂O₂. Sections were further blocked with CAS-block reagent (Invitrogen, Waltham, MA, USA) for an hour to prevent nonspecific binding, and exposed with primary antibody including Ki67 (Abcam, 1:100) and c-casp3 (Abcam, 1:100) at 4 °C overnight, and with the secondary antibody at room temperature for 1 h. Chromogen reaction with DAB (Abcam, Cambridge, UK) was then performed to visualize the presence of HRP/peroxidase, and the slides were counterstained with hematoxylin. Live images were taken under the light microscope, and the presence of Ki67 and c-casp3 within colorectal tumor was quantified with the ImageJ software (NIH, USA). The histoscore system was adopted based on Fedchenko et al. [2 (link)]. The histoscore of IHC was given by multiplying the percentage of positively stained area with the intensity graded from 0, non-stained; 1, weakly stained; 2, moderately stained; and 3, strongly stained.

Tissue samples were cut and embedded in paraffin, and then baked, dewaxed, and hydrated. Antigen retrieval was performed by dropwise addition of 3% H₂O₂. After blocking non-specific binding with 5% BSA, the samples were treated with the appropriately diluted primary antibody overnight at 4 °C. The samples were then washed and incubated for 1 h with the appropriate biotinylated secondary antibody, and then SABC (SA1022, Boster Bio Engineering Company, Wuhan, China) was added dropwise. Samples were developed with DAB (59,718, Abcam) and examined by light microscopy. The final hematoxylin staining was terminated with ddH₂O. Antibodies against CDK2 (Proteintech, 10,122–1-AP, 1:250), PHIP (Bioworld, BS8775, 1:100), DNMT3B (Proteintech, 26,971–1-AP, 1:250), and Sp1 (Cell Signaling Technology, 9389, 1:2000) were used for IHC.

The hepatic tissue fixed in 10% neutral buffered formalin was embedded in paraffin and cut into 5 μm thick sections for histomorphological and immunohistochemical examination. After drying, hepatic tissue section slides were stained with hematoxylin and eosin (H&E) according to standard procedures. For immunohistochemistry, sections were incubated with 4-hydroxynonenal (4-HNE) primary antibody (1 : 200; Abcam, Cambridge, UK) and biotinylated secondary antibody (Nichirei Biosciences, Tokyo, Japan), followed by the avidin-biotin-peroxidase complex. The immunoreactive signal was developed by their substrates, DAB (3,3′-diaminobenzidine) and AEC (3-amino-9-ethylcarbazole) (Abcam). The slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO) and examined under an optical microscope (Leica Microsystems, Wetzlar, Germany).

The lung tissue sections were deparaffinized and subjected to microwave-mediated antigen retrieval in citrate buffer (pH = 6.0), prior to blocking with 1% BSA for 30 min at 25 °C. IL-10 or IL-6 antibody (Santa Cruz Biotechnology) was diluted 1:100 and incubated with the sections overnight at 4 °C. HRP polymer-conjugated goat anti-mouse secondary antibody and DAB (Abcam, Cambridge, MA) were applied to indicate the positive area59 (link).

The samples of NPC tissue arrays or syngeneic tumor were subjected to heat-mediated antigen retrieval in 0.01 mol/l citrate buffer (pH 6.0). After cooling to room temperature, samples were treated successively with 1% methanol/30% H₂O₂ to block endogenous peroxidase and 5% bovine serum albumin to block nonspecific binding sites. After rinsing with PBS, they were incubated overnight at 4°C with the goat-anti-TNFα or rat-anti-CD68 primary antibody (Abcam, Cambridge, UK). The sections were then rinsed with PBS and incubated with the HRP conjugated secondary antibody (Abcam, Cambridge, UK) for 30 min at room temperature. Then the sections were washed and incubated with DAB (Abcam, Cambridge, UK), and was terminated by rinsing with distilled H₂O. Finally, the samples were counterstained with hematoxylin.

Patient GBM samples were collected, processed and stored by the Leeds Multidisciplinary Routine Tissue Banking (RTB) service, obtained from GBM patients undergoing surgery at the Leeds General Infirmary (ethical approval no. RTB 15/YH/0080). Tumour tissues were embedded in paraffin wax, sectioned on a microtome (Leica Biosystems, Newcastle Upon Tyne, UK) at 8 µm and dried on glass microslides for 24 h. The slides were deparaffinized and rehydrated following standard procedures, then dewaxed by 2× 1-min washes in xylene (Sigma, Poole, Dorset, UK), followed by 2× 1 min washes in ethanol (Sigma, Poole, Dorset, UK) and 3× 1 min washes in water. Antigen retrieval was performed using Tris EDTA, pH 9 (Abcam, Cambridge, UK) in a pressure cooker for 2 min. The primary antibody anti-CYR61/CCN1 rabbit polyclonal (Abcam, Cambridge, UK) was used at a concentration of 1:250. A secondary antibody (anti-rabbit IgG HRP polymer (ready to use, Vector, 2BScientific, Upper Heyford, UK)) was used for signal amplification and DAB (Abcam, Cambridge, UK) reaction (10 min) was used for signal detection. Slide analysis was carried out by calculating and combining two scores for the CCN1 staining as described above for TMA analysis. The samples were randomised prior to scoring to prevent bias.