Donkey serum

Methods for immunostaining have been described previously^{66 (link),121 (link),122 (link)}. Cells were fixed for 20 min at 4 °C in PBS 4% PFA (electron microscopy grade), rinsed three times with PBS, and blocked and permeabilized at the same time for 30 min at room temperature using PBS with 10% Donkey Serum (Biorad) and 0.1% Triton X-100 (Sigma). Incubation with primary antibodies diluted in PBS 1% Donkey Serum 0.1% Triton X-100 was performed overnight at 4 °C. Samples were washed three times with PBS, and then incubated with AlexaFluor secondary antibodies for 1 h at room temperature protected from light. Cells were finally washed three times with PBS, and Hoechst (Sigma) was added to the first wash to stain nuclei. Images were acquired using an LSM 700 confocal microscope (Leica).

The grafted skin tissues were embedded in Tissue-Tek® Optimal Cutting Temperature compound (Sakura Fineteck, Tokyo, Japan) and sectioned at 10-μm thickness. The sections were washed thrice with Tris-buffered saline (TBS, TaKaRa Bio, Shiga, Japan) for 10 min and blocked for 1 h in TBS (TaKaRa Bio) containing 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and 0.3% Triton X-100 (FUJIFILM Holdings Corporation, Tokyo, Japan). The sections were then probed overnight at 4 °C with anti-CD3 (CD3-12; Abcam, Cambridge, UK, USA) and anti-CD45 (L12/201; Bio-Rad) antibodies in TBS containing 1% donkey serum and 0.3% Triton X-100. Subsequently, samples were again washed thrice with TBS for 5 min and were labelled for 1 hour at room temperature (20 °C–28 °C) with secondary antibodies conjugated with Alexa Flour 488 (Thermo Fisher Scientific). The sections were counterstained with Hoechst 33342 (Thermo Fisher Scientific) to visualise the nuclei and imaged using a fluorescence microscope (Axio Observer. D1, Carl Zeiss, Jena, Germany).

The cells were fixed in 4% paraformaldehyde for 20 min at 4 °C followed by one wash with PBS. The cells were subsequently blocked and permeabilized at room temperature for 30 min in PBS with 4% donkey serum (Bio-Rad) and 0.1% Triton X-100 (Sigma–Aldrich). Primary antibodies were diluted in the same buffer and incubated with the cells overnight at 4 °C. After three washes with PBS, the cells were incubated with Alexa Fluor secondary antibodies for 1 h at room temperature protected from light. The cells were subsequently washed three times for 5 min with PBS, adding 5 μg/ml DAPI during the first wash to stain nuclei. The antibodies used are listed in Table S3.

The immunostaining method has been described previously^{18 (link),25 (link),73 (link)}. Cells were fixed for 20 min at 4 °C in PBS 4% PFA (electron microscopy grade), rinsed three times with PBS, and blocked and permeabilised at the same time for 30 min at room temperature using PBS with 10% Donkey Serum (Biorad) and 0.1% Triton X-100 (Sigma). Incubation with primary antibodies (Supplementary Information—Supplementary Table 1) diluted in PBS 1% Donkey Serum 0.1% Triton X-100 was performed overnight at 4 °C. Samples were washed three times with PBS, and then incubated with Alexa Fluor secondary antibodies (Supplementary Information—Supplementary Table 1) for 1 h at room temperature protected from light. Cells were finally washed three times with PBS, and Hoechst (Sigma) was added to the first wash to stain nuclei. Images were acquired using an LSM 700 confocal microscope (Leica).

Organoids were treated overnight with 15 µM Mitomycin C (Sigma, M4287). About 16 h later, organoids were harvested and fixed in 4% formalin overnight at 4 °C. Prior to the whole-mount staining, the fixed organoids were permeabilized with 0.5% Triton-X (Sigma), 2% donkey serum (BioRad) in PBS for 30 min at 4 °C and blocked with 0.1% Tween-20 (Sigma) and 2% donkey serum in PBS for 15 min at room temperature. Subsequently, organoids were stained with mouse anti-γH2A.X primary antibody (1:500, Millipore, clone JBW301) overnight at 4 °C, followed by four washes with PBS and incubation with secondary goat anti-mouse AF-647 antibody (1:250, Thermo Fisher, catalog number A-21235) for 2 h at room temperature in the dark and washed again with PBS. DAPI was used to counterstain nuclei. Organoids were mounted and imaged on an SP8 confocal microscope (Leica). Fluorescent microscopic images of γH2A.X were quantified as follows: Based on the staining, the nuclei were classified as γH2A.X-positive or -negative. The fraction of positively stained nuclei over all nuclei is displayed as one datapoint per organoid. At least 10 organoids were quantified per line over two independent experiments.