Vectashield mounting media containing dapi

Organoids were seeded out in 4- or 8-well chamber slides (Thermo Fisher Scientific) before use in stimulations and microscopy. Organoids were stimulated for 48 h before use. For immunofluorescence staining, cultured organoids were washed in PBS twice and then fixed in 4% paraformaldehyde for 20 min at room temperature. Organoids were then permeabilized with PBS containing 0.5% Triton X-100 for 10 min at 4°C, followed by rinsing three times with PBS containing 100 mM glycine. Organoids were blocked with IF buffer (PBS containing 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20, and 10% FCS) for 1 h at room temperature before incubation with 1/1,000 anti-mouse Dclk1 (Abcam) in antibody diluent (Invitrogen) at 4°C overnight. After rinsing three times with IF buffer, slides were incubated with anti-rabbit FITC (Dako) for 1 h at room temperature. Slides were then washed with IF buffer followed by three rinses with PBS before being mounted with Vectashield mounting media containing DAPI (Vector Laboratories). Slides were imaged using a Leica DMi8 inverted microscope and Leica Application Suite (LAS) X software (Leica Microsystems). Resulting image files were analyzed using ImageJ/Fiji (Schindelin et al., 2012 (link)).

For immunocytochemistry of NPCs, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After blocking nonspecific staining in PBS containing 10% normal goat serum (NGS), 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 45 min at RT, cells were incubated with the primary antibodies overnight at 4°C. Primary antibodies were diluted in PBS containing 1% NGS, 1% BSA and 0.1% Triton X-100. All primary antibodies used in the study are commercially available and well characterized. We used primary antibodies recognizing SOX2 (1:25, MAB 2018; R&D Systems), Nestin (1:200, SC-20978; Santa Cruz Biotechnologies), DCX (1:500, AB2253; Millipore), and MAP2 (1:2500, M4403; Sigma). Secondary antibodies were applied in PBS containing 1% BSA for 45 min at RT. Secondary antibodies were goat anti-chicken Alexa Fluor 488 (1:500, A-1139, Invitrogen), anti-rabbit Alexa Fluor 546 (1:500, A11003; Invitrogen), and anti-mouse Alexa Fluor 635 (1:500, A-31574; Invitrogen). After final washes, the cell nuclei were counterstained with Vectashield mounting media containing DAPI (Vector Laboratories).

For in situ analysis of DSB repair by HR, we performed immunofluorescence microscopic analysis of the DNA damage marker γH2AX and the recombinase RAD51 in PBLs after exposure to 2 Gy of ionizing radiation (IR) as previously described (Gatz et al., 2016; Obermeier et al., 2016). Briefly, PBLs were harvested by cytospinning at the indicated time points postirradiation, fixed with 3.7% formaldehyde followed by permeabilization with 0.5% TritionX‐100. Primary antibodies were directed against γH2AX (Ser139, clone JBW301, Millipore, Billerica, MA, USA) and RAD51 (H‐92, Santa Cruz Biotechnology, Heidelberg, Germany), and the secondary antibodies were AlexaFluor488 and 555‐labeled (Invitrogen, Karlsruhe, Germany). Immunostained cells were mounted with VectaShield mounting media containing DAPI (Vector laboratories, Burlingame, CA, USA). Focal accumulations of 53BP1 and RAD51 in ≥97 DAPI‐stained nuclei from two independent slides were analyzed using a Keyence BZ‐9000 microscope equipped with Keyence BZ‐II Analyzer software (Keyence, Neu‐Isenburg, Germany).

For RAD51 nuclear focus formation analysis, cells were cultured on coverslips for 24 h and then irradiated or not with 4 Gy using a RS2000 generator (Radsource). After irradiation, cells were allowed to recover for 4 h. Cells were then washed with PBS, treated with CSK buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 0.7% Triton X-100) for 2 min at 4°C, washed with PBS and fixed with 4% paraformaldehyde for 10 min at 4°C before an additional fixation step of 2 min with glacial methanol. Coverslips were rinsed with PBS and blocked for 1 h in PBS containing 0.1% Triton X-100 and 5% BSA. Cells were stained using a rabbit anti-RAD51 serum (1:5000) [106 (link)] in 0.5% BSA PBS at room temperature for 1 h prior to incubation with AlexaFluor 594 or 488 anti-rabbit secondary antibodies (1:5000, Molecular Probes) in 0.5% BSA PBS. Coverslips were mounted onto slides with Vectashield mounting media containing DAPI (Vector laboratories) and images were obtained using a Zeiss Axio Imager Z2 microscope with a 63x oil immersion objective. Maximum-intensity projection images were generated to display foci in all sections and were analyzed using ImageJ software. Automatic counting was performed using FoCo [107 (link)] and validated manually.

Mice were generated in which exons 4 and 5 of Kindlin1 (Fermt1) were floxed (Kin1^fl/fl) (Taconics). Kin1^fl/fl mice were interbred with K14CreER^T2 (K14Cre) mice (Li et al., 2000 (link)) to give experimental cohorts of K14Cre and K14Cre Kin1^fl/fl mice on a FVB/N background. Genotyping was carried out by Transnetyx. All experiments were carried out in accordance with the UK Animal Scientific Procedures Act (1986). Eight-week-old mice were injected with 0.1 mg 4-hydroxytamoxifen (4OHT) (Sigma) for 5 days. After a further 10 days, animals were sacrificed, and dorsal skin was removed and fixed in 10% neutral buffered formalin. Sections were deparaffinized, rehydrated, and unmasked in 10 mM citric acid (pH 6.0) in a pressure cooker for 10 min. They were then blocked with peroxidase (15 min) and protein blocking buffers (2 h) at room temperature (DakoCytomation). Sections were incubated overnight at 4°C with primary antibodies diluted in Antibody Diluent, washed 5 times in 0.1% Tween-20/TBS (TBST), and incubated with Alexa Fluor-488-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Sections were rinsed 5 times in TBS and mounted with Vectashield Mounting Media containing DAPI (Vector Laboratories). The following antibody dilutions were used: anti-alpha-tubulin, 1:300 (CST, 2125) and anti-acetylated-tubulin, 1:800 (CST, 12152)