Biotin conjugated donkey anti rabbit

Tissues were fixed in 10% formalin overnight, paraffin embedded, and sectioned. Deparaffinized sections were subjected to antigen retrieval using Borg Decloaker RTU solution (Biocare Medical) in a pressure cooker (Instant Pot) for 20 minutes. Sections were incubated with rabbit anti-Ki-67 (D3B5) primary antibody (1:400, CST 12202S) overnight at 4°C. Secondary antibody used was biotin-conjugated donkey anti-rabbit (Jackson ImmunoResearch), followed by detection using the Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs) and Dako Liquid DAB+ Substrate (Dako). Sections were counterstained with hematoxylin and mounted with Cytoseal XYL (Thermo) for visualization. Antibody dilutions were made in Common Antibody Diluent (BioGenex). Number of crypts with >10 Ki-67 positive cells were averaged across 0.5 cm of tissue.

Three wild-type mice at postnatal day five from the albino C57BL/6J-Tyr^c-Brd inbred strain were used for the expression analysis. The heads of all samples were dissected in PBS before fixation for two days in 10% formalin at 4°C, washing, dehydrating and embedding in paraffin wax. Embedded samples were cut into 8μm thick sections along the sagittal plane. Immunohistochemistry was then carried out according to the manufacturer’s instructions on slides using the Ventana Discovery machine with the manufacturer’s reagents CC1 (cat.no 950–124), EZPrep (cat.no 950–100), LCS (cat.no 650–010), RiboWash (cat.no 760–105), Reaction Buffer (cat.no 95–300), and RiboCC (cat.no 760–107). The DABMap Kit (Ventana; cat.no 760–124) with hematoxylin counterstain (cat.no 760–2021) and bluing reagent (cat.no 760–2037) were used. All antibodies were diluted in ‘Antibody staining solution’: 10% fetal calf serum, 0.1% Triton, 2% BSA and 0.5% sodium azide in PBS. The primary antibodies used were anti-CD164 (SantaCruz, sc-33124, 1:75 and St.John’s Laboratory, STJ92095, 1:500). The secondary antibody used was Jackson ImmunoResearch biotin-conjugated donkey anti-rabbit (711-065-152, 1:100). The stained slides were examined and images obtained using an AxioCam HRc camera mounted on a Zeiss microscope.

Mouse paw tissue paraffin embedded sections from 7 month old COMP deficient 129/Sv mice^{32 (link)} and age matched WT mice were analysed. Paraffin removal and then antigen retrieval were obtained by incubation in an alkaline solution (K8004 EnVision FLEX Target Retrieval Solution, High pH 9.0, DAKO, Denmark A/S, Glostrup, Denmark) at 85 °C for 40 min. Sections were cooled in washing buffer (K8007 EnVision FLEX Wash Buffer, DAKO) and blocked for 1 h at room temperature (RT) with 2% normal donkey serum in PBS (Jackson ImmunoResearch Laboratories, INC., West Grove, PA), also used for antibody dilution. Primary rabbit anti-lubricin antibody (P3-118, Thermo Scientific) at 2 µg/ml was incubated overnight at 4 °C followed by incubation with the secondary antibody for 1 h at RT with 3 × 5 min intermediary washes with washing buffer. The secondary antibody was either Rhodamine Red-X-conjugated, donkey anti-rabbit (711-296-162, Jackson ImmunoResearch, diluted 1 in 1000), or biotin-conjugated, donkey anti-rabbit (Jackson ImmunoResearch, diluted 1 in 1000) visualised after incubation with streptavidin-FITC for 30 min at RT. Negative control was performed with primary antibody omitted. Finally, sections were mounted as above and images were captured in a Zeiss Axioscope 2 Plus fluorescence microscope and images were cropped and contrast adjusted for the complete image in Adobe Photoshop CC.