Biotinylated goat anti rabbit secondary antibody

Femurs from the aged and young C57Bl/6 mice were cleaned from adherent tissue and maintained in EDTA solution (0.5 M, pH: 7.2–7.4) on a shaker. When a complete decalcification process was reached, the femurs were washed three times in PBS and included in paraffin using a standard histological technique. Five-micrometer serial sections were cut and used for immunohistochemistry studies. To detect the presence of iNOS-positive cells, the sections were treated with an anti-iNOS antibody (ab15323) (Abcam, Cambridge, UK). A heat mediated antigen retrieval was performed in a microwave with sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) and left at RT for 20 min. Non-specific sites were blocked with a solution of 10% NGS, 1% BSA (Sigma, Darmstadt, Germany) in TBS at RT for 15 min. The slides were incubated with the primary antibody diluted (1:100) in TBS-1% BSA overnight at 4 °C and then detected using a biotinylated goat anti-rabbit secondary antibody (Dako Cytomation, Milano, Italy). Negative controls with pre-immune serum were run in parallel. The images were taken by Leica DMi1 microscope (Leica Microsystems, Milano, Italy).

Frozen GCT were blocked with OCT and 4 μm sections were prepared using the cryostat. All incubations and washes were performed at room temperature unless stated otherwise. Frozen GCT sections were fixed in 4% paraformaldehyde for 30 min followed by quenching of endogenous peroxidase using 0.3% hydrogen peroxide/PBS for 30 min. For membrane permeation, slides were incubated in 0.1% triton X-100/PBS for 10 min. Nonspecific binding was blocked by 10% goat serum in 3% BSA for an hour. Incubations with primary antibody, rabbit polyclonal INSL3 (Abcam ab 65981; 1:250), CXCL14 (Abcam ab46010; 1:400) or MFAP5 (Sigma abHPA010552; 1:500) was performed at 4°C overnight. Goat serum was used as a negative control. After PBS washes, slides were incubated with biotinylated goat anti-rabbit secondary antibody (Dako; 1:200) for an hour. VECTORSTAIN® avidin/biotinylated enzyme complex was made up as per manufacturer's instructions, added to sections and incubated for an hour. Staining was visualised by incubation of DAB solution (Dako) for 3 min. Sections were counterstained with hematoxylin, dehydrated with ethanol (70% and 100%) and mounted with DPX. Validation of the antibodies using a positive control tissue is shown in Supplementary Figure 2.

Specimens were collected from patients with advanced NSCLC and activating EGFR mutations, all patients had already accepted gefitinib as first-line treatment between January 2013 and December 2015 at Daping Hospital of the Third Military Medical University (Chongqing, China). All the tissue section slides were baked at 60°C for 2hr, followed by deparaffinized and hydrated, and incubated with 3% hydrogen peroxide. After being microwaved in 0.01M sodium citrate buffer for 30min, the sections were preincubated in 10% normal goat serum for 30 min to prevent nonspecific staining. Subsequently, the sections were incubated with the BIM polyclonal antibody overnight at 4°C. Sections were then sequentially incubated with biotinylated goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) for 30 min each at room temperature. The immunostaining results were substantiated independently by two experienced pathologists blinded to the patient’s identity and clinical status. Scoring for IHC staining was based on the criteria described previously [47 (link)]. The study was approved by the Ethics Committee of Daping Hospital of the Third Military Medical University. Informed consent was obtained from all individual patients or relatives included in the study.