Immpact vector red

Directly after µCT imaging porcine osteochondral samples were fixed for 72 h in 4% formaldehyde and decalcified for 13 to 15 days in Formical^® 2000 (StatLab, McKinney, TX, USA). Subsequently, specimens were dehydrated, PCL remnants dissolved via Xylene as intermedium, and samples embedded in paraffin. After cutting off 5 µm serial sections, slides were stained with Safranin O/Fast Green according to standard protocols. Type II collagens were stained with mouse anti-human monoclonal antibodies (MPBiomedicals, Eschwege, Germany; Jackson ImmunoResearch, Ely, United Kingdom) according to standard immune histology protocols with ImmPact Vector Red counterstaining (Vector Laboratories, Burlingame, CA, USA).

Paraffin sections of testes were prepared as above at 7 µm in thickness, and de-paraffinized and hydrophilized with xylene, ethanol, and Milli-Q water. The sections were boiled for 45 min with 10 mM sodium citrate-HCl (pH 6.0) and treated with 3% H₂O₂-containing PBS. After the H₂O₂ treatment, the blocking was performed by soaking the sections with 1% bovine serum albumin in 10 mM Tris-HCl (pH 7.5), 0.88% NaCl, and 0.1% Tween20 for 1 hr at room temperature. An anti-HSD3B antibody (no dilution; Cat# sc-515120, Santa Cruz Biotechnology, Dallas, TX) was reacted with the section for 1 hr at room temperature. After washing with PBS, the sections were subjected to the 2nd antibody reaction for 1 hr at room temperature with a HRP-Labelled Polymer Anti-Mouse (1:4 dilution, Dako). After washing with PBS, the sections were stained with ImmPACT Vector Red (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions.

Lung tissues from two animals per treatment group and time point were processed and stained as previously described (9 (link)). Antibodies specific for PR8 hemagglutinin protein (catalog number NR-3148; BEI Resources) and Ly6G (clone 1A8; Bio X Cell) were detected with immPACT vector red and diaminobenzidine immPACT DAB, respectively (Vector Laboratories).

Directly after micro-CT imaging, porcine osteochondral samples were fixed for 72 h in 4% formaldehyde followed by either decalcification for up to 16 weeks in ethylenediaminetetraacetic acid or 13–15 days in Formical® 2000 (StatLab, USA) (n = 30), respectively, or processed for polymethylmethacrylate (PMMA) embedding (n = 46). Decalcified specimens were dehydrated and dissolved in acetone and xylene for PCL remnants, before samples were embedded in paraffin. After cutting 5 μm serial sections, type II collagens were stained with mouse anti-human monoclonal antibodies (MP Biomedicals, Germany; Jackson ImmunoResearch, UK) according to standard immune histology protocols with ImmPACT Vector Red counterstaining (Vector Laboratories, USA).
PMMA embedded specimens were stained using toluidine blue. Polymerization was completed in an incubator at 37.5°C. Ground sections were cut, mounted on Acropal slides and polished before surface staining with toluidine blue.

Formalin-fixed paraffin-embedded tissue obtained from surgical resection of patient tumours was used for IHC analysis. Tumours were processed using an automated Bond Max system (Leica Biosystems, Milton Keynes, UK) as described [35 (link)]. Mouse-anti-human CD8 antibody (Dako) was used at 1:100 dilution, followed by anti-mouse secondary (Abcam, Cambridge, UK) at 1:500; CD8 positivity was detected using ImmPACT Vector Red (Vector Labs, Oxfordshire, UK). Control sections were processed without the addition of primary antibody. Digital images were acquired at ×20 magnification and quantified using ImageScope software (version 12.4.3.5008, Leica Biosystems).