Anti rabbit secondary antibody

siRNA assays samples were electroblotted onto a Hybond-C Extra membrane (Amersham, Chalfont St Giles, UK), and antibody recognition was performed using in-house or commercial antibodies. A blocking solution was added to cover the membrane: the buffer contained 3–5% lyophilised milk for alimentary use, 1X PBS and 0.1% Tween20. The membrane was incubated for at least one hour and washed again with 1X PBS. The membrane was consequently incubated with the specific primary antibody, diluted in 1X PBS + 0.1% Tween20 and 3% BSA, for 1–2 hours. The membrane was washed with 1X PBS three times, and incubated with the appropriate secondary antibodies for 1–2 hours and washed again. Protein bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The following antibodies were used in this study: Polyclonal Anti-PTB antibody produced in rabbit (in house from ICGEB, Trieste, Italy); dilution: 1:1000. Polyclonal Anti-hnRNP H antibody produced in rabbit (SIGMA; catalogue number: SAB4501422); dilution: 1:500 Anti-Calnexin antibody (Abcam; catalogue number: ab22595); dilution: 1:1000. Anti-Rabbit secondary antibody (Dako); dilution: 1:2000.

Formalin-fixed tissue was processed for routine histological examination. 4 μm sections were stained with Hematoxylin–Eosin (HE) and examined by a pathologist (NCMV) for typing and grading of the tumours. To verify presence of human cells, IHC staining was performed on full section to detect expression of human ERα. Briefly, sections were dewaxed with xylene, rehydrated in graded ethanol before microwave antigen retrieval, and stained for 60 min in room temperature for human ERα expression using 1:400 HC-20 (Santa Cruz Biotechnology, Dallas, TX, USA) or 1:400 Clone SP1 (Thermo Scientific, Fremont, CA, USA). Anti-rabbit secondary antibody (Dako, Denmark) was applied for 30 minutes, followed by 8 minutes with Diaminobenzidine (DAB+, Dako, Denmark) before counterstaining with hematoxylin. To verify the species specific nature of the antibody, sections were compared to the non-species specific antibody from Santa Cruz (S1 Fig), validating that the human specific antibody only detected ERα in cells of human origin.

Kidney grafts were collected from the recipient mice, and embedded in OCT (optimal cutting temperature) compound (Tissue-Tek, Sakura Finetek, CA). Sections were cut at 7 µm thickness, and blocked with 0.3% hydrogen peroxide.
To measure graft volume, every 6^th section was stained for insulin using rabbit anti-insulin antibody (Cell Signaling, Danvers, MA), anti-rabbit secondary antibody (DAKO, Carpinteria, CA), a peroxidase substrate containing 3,3-diaminobenzidine (DAB-brown) in a DAKO IHC autostainer. Sections were counterstained with hematoxylin on a Leica autostainer XL before mounting with MM 24 (Leica Biosystems, Nussloch, Germany) and a Leica auto-coverslip machine CV5030. Images were taken with a Leica DFC 450 camera on a Leica DM 4000 microscope.

For histological analysis, spleen and tumor tissues were cut into 5 µm sections, deparaffinized with xylene, and hydrated with graded ethanol. Endogenous peroxidase was blocked using 3% H₂O₂ for 5 min. and 0.01M citrate buffer (pH 6.0) was used for antigen retrieval. The samples were cooked in a pressure cooker at maximum power for 13 min and then at 40% power for an additional 15 min, and left to cool for 30 min until they reached room temperature. All slides were then washed in PBS and blocked for 30 min with CAS block at room temperature. The tissues were incubated with rabbit anti-CD4 Ab (ab183685) or rabbit anti-CD8+ Ab (ab203035) diluted in CAS-Block (1:1000 or 1:250, respectively) overnight at 4°C. The following day, the slides were washed in PBS, incubated with Dako anti-rabbit secondary antibody for 30 min and developed with AEC for 10 min. After several washes in PBS and dH₂O, the slides were counterstained with hematoxylin, rinsed in H₂O, and covered with fluoromount (ThermoFisher) and a cover-slip. The photos were taken with an Olympus BX50 microscope and Olympus DP73 camera at room temperature. The acquisition software used was CellSens Entry 1.8.