Protein block

To analyse the neovascularisation within transplanted PEG hydrogels, paraffin-sectioned slides were stained for mouse endothelial cells. First, sections were deparaffinised with Xylene and rehydrated in 100% ethanol. Slides were incubated in 3% H₂O₂ for 30 min at room temperature to block any endogenous peroxidase activity. Then, slides were incubated in 10 mmol/l Tris EDTA (pH 9) solution for 20 min at 96 °C and additional 20 min at room temperature for antigen retrieval. Afterwards, non-specific bindings were blocked by the protein block (Abcam, Cambridge, UK) for 1 h followed by overnight incubation at 4 °C with primary antibodies—rabbit anti-mouse CD34 antibody (1:500 dilution; Abcam, catalog#ab81289) dissolved in Tris-buffered saline with 1% Normal Rabbit Serum (Sigma-Aldrich) and 0.1% bovine serum albumin (Fisher Scientific, Waltham, MA, USA). Following incubation overnight, slides were treated for 20 min each with the biotinylated goat anti-polyvalent and streptavidin peroxidase, provided in Rabbit Specific HRP/DAP Detection IHC kit (Abcam). Haematoxylin was used for counterstaining. Healthy mouse uterus was used as positive-control and negative-control slides were incubated without the presence of primary antibody (Supplementary Figure S3). The identical procedure was followed for both positive and negative control as described.

The embryonic testes were dissected from 13.5 embryos and fixed in 4% formaldehyde in PBS. We prepared paraffin-embedded tissue section (5μM thick). The primordial germ cells were labelled by anti-SSEA1 antibody (ab16285) (Abcam,). In brief the tissue sections were deparaffinized, rehydrated and incubated in PBS for 5 min. The antigen retrieval was performed by heating the slides with sections in citrate buffer (IHC world general protocol) and HistoReveal (Abcam) according to the manufacturer protocol (5 min incubation at RT) and Protein Blocking was performed by Protein Block (Abcam) during 10 min at RT. The slides were subsequently incubated with the primary antibody (anti-SSEA1, Abcam, ab16285, 10 μg/ml) at 37°C for 60 min. After washing in PBS, slides were incubated with the secondary antibody (Goat Anti-Mouse IgM H&L, Abcam, 10 μg/ml) at 37°C for 60 min. After washing in PBS and water, the tissue slides were used for TUNEL assay according to the protocol described above with some modifications. In these samples no formalin fixation and proteinase K digestion were performed. Subsequently, slides were mounted with Vectashield (Vector) (DAPI counterstaining) and analysed under a fluorescent microscope. The numbers of TUNEL-positive and SSEA1-positive cells were counted with NIS Elements picture analyser and normalized to the sample area.

Immunohistochemical staining, for IL-6, CCL21 and for CD4+ and CD8+ cells, was performed in dewaxed paraffin sections. Sections were dewaxed in Histochoice (VWR, Visalia, CA, USA), passed through graded alcohols, and rehydrated with water. Then, antigen retrieval was performed by incubating sections in bowled citrate buffer for 15 min at room temperature. After blocking with Protein Block (Abcam kit, Burlingame, CA, USA), sections were incubated overnight with rabbit anti-rat polyclonal IL-6, CD4-antibody (Novus Biological, CO, USA), CCL21-antibody (Invitrogen/Thermo Fisher, Scientific, Waltham, MA, USA) or mouse anti-rat monoclonal CD8 antibody (Thermo Fisher) at 4°C. Then, the sections were treated with hydrogen peroxide blocking reagent at room temperature and were processed using EXPOSE Mouse and Rabbit specific HRP/DAB Detection IHC Kit (Abcam kit, cat#80436) according to the manufacturers’ instructions, followed by counterstaining with Mayer’s hematoxylin solution (IHC World LLC, Ellicott city, MD, USA). As negative control, 2.5% normal goat serum was used to confirm specific staining of secondary antibodies. Stained sections were examined by light microscopy. Data was quantitated using ImagePro.Plus software. For quantification, 3 sections at 100 μm interval were analyzed from each knee, and for CD4 and CD8, we focused on synovial tissue at the joint capsule.

3 μm sections were cut from paraffin-embedded tumor tissue. After de-paraffinization, antigen retrieval was carried out by incubating sections in Tris EDTA buffer for 20 mins at 95°C. Endogenous peroxidase activity was blocked by incubation in “Peroxidase Block” (Abcam) for 10 mins. Sections were washed and then incubated in “Protein Block” (Abcam) for 20 mins. Antibodies against IL-6, KC, Survivin, BCL-XL, Versican, TNF-α and VEGF (Abcam) were incubated on individual slides at 4°C for 16 hrs in a moist chamber. An ABC (Avidin Biotin conjugate) kit (Abcam) was employed to visualize reactivity. Images (40X) were captured on a Leica microscope using a Leica ICC50W camera and processed using LAS V4-10 software.

After treatment, mice were euthanized and dissected. Fresh mouse DRG samples were isolated and immediately fixed with formalin. Paraffin-embedded DRG tissue sections were prepared, and slides were processed for IHC staining as previously described.^{14 (link)} In brief, sections were deparaffinized and rehydrated. They were then pretreated with Target Retrieval Solution, pH 6.0 (DAKO). Sections were blocked with Protein Block (Abcam) and then incubated with primary antibody against SIRT2 (Proteintech), Acetyl-α-Tubulin (Cell Signalling), and anti-NeuN antibody (Abcam). After overnight incubation with primary antibodies, slides were washed and incubated with secondary goat anti-mouse Alexa Fluor 594-conjugated antibodies or goat anti-rabbit Alexa Fluor 488-conjugated antibodies (Thermo Fisher Scientific) and analyzed by fluorescence microscopy (Carl Zeiss). Immunoreactivity was quantified using ImageJ, and expressions from each mouse DRG were plotted.