Zen blue

For analysis, the labeled sections were examined with an Axio Imager.M2 equipped with an ApoTome.2 module or a Laser Scanning Microscope 710 with corresponding imaging modules (Carl Zeiss AG, Oberkochen, Germany). Images were acquired using a 20 × (0.8 NA, Apochromat) or a 63 × (1.4 NA oil immersion, Plan Apochromat) objective (both Carl Zeiss AG) as stacks of multiple optical sections and projections were calculated with ZEN blue or ZEN black software (Carl Zeiss AG). Images were adjusted for contrast and brightness using Photoshop CS6 (Adobe Systems, San Jose, CA, USA) and arranged using CorelDRAW (Corel Corporation, Ottawa, ON, Canada). For quantifying the colocalization experiments, three animals of each genotype were analyzed. Per animal, three images were acquired at different regions of the retina and cell counting was performed using ZEN blue (Carl Zeiss AG). Cell numbers were summarized in Excel (Microsoft Corporation, Redmond, WA, USA) and graphs were created using GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, CA, USA). Graphs and images were arranged using CorelDRAW (Corel Corporation).

Skin, lung, liver, and kidney were dissected and fixed in 4% formaldehyde for 24 h. Paraffin sections were stained with Hematoxylin‐Eosin (H&E) to evaluate inflammation in these organs. Images were acquired using Zeiss Axioscan and Zen Blue software.
Murine gut was dissected and fixed in 4% formaldehyde for 24 h. Paraffin sections were stained with Hematoxylin‐Eosin (H&E) and ileal TNF^emARE sections were scored blindly by assessing villus architectural distortion (0–4), goblet cell depletion (0–4), and mononuclear cell infiltration (0–4) resulting in an overall score of 0–12 (Appendix Table S1). Images were acquired using Zeiss Axioscan and Zen Blue software.
Murine paws and spine were dissected, fixed in 4% formaldehyde for 48 h and then decalcified using 5% formic acid for 8 consecutive days. Paraffin sections were stained with H&E for evaluation of inflammation and bone erosions. Disease development in hind paws was scored blindly by assessing the parameters in Appendix Table S2, based on Yang–Hamilton (Yang & Hamilton, 2001 (link)) scoring and SKG scoring (Ruutu et al, 2012 (link)). To quantify inflammation in the spine, H&E sections were scored for the extent of immune cell infiltration (0–3) along the longitudinal ligament and in the intervertebral discs at the thoracic segment (Appendix Table S3). Images were acquired using Zeiss Axioscan and Zen Blue software.

For immunofluorescence experiments, slides were imaged on a Zeiss Axio Imager epifluorescence microscope at 63x magnification. DAPI, AF488, AF594, and AF647 were imaged sequentially using standardized exposure times for each antibody condition and processed using Zeiss Zen Blue version 3.0 (Carl Zeiss AG, Oberkochen, Germany). Images were adjusted in ImageJ (National Institutes of Health, USA) to standardize background across all images. Diakinesis cells stained with Giemsa were imaged on a Zeiss Axio Imager epifluorescence microscope at 40x magnification using brightfield. Images were processed using Zeiss Zen Blue version 3.0 (Carl Zeiss AG, Oberkochen, Germany). In most cases, SYCP3 was used to sub-stage cells, with the extent of XY synapsis used to substage pachytene into early, mid and late. For FANCJ colocalization studies with TOPBP1, MSH4, MLH1, and BRCA1, cells were sub-staged by DAPI. Leptonema/zygonema were defined by 21–40 centromeres (DAPI-dense spots) without a visible sex body; pachynema having 21 centromeres and a defined sex body; and diplonema having more than 21 centromeres and a defined sex body. Zygotene cells could be further categorized by early/mid and late using FANCJ staining.

Spheroids were visualized with phase contrast microscopy in an appropriate culture medium (n = 4, Zeiss Axiovert 25, 10× objective; Carl Zeiss Microscopy Deutschland, Oberkochen, Germany) after initial spheroid formation and after 14 and 28 days under unstimulated and osteogenic cultivation conditions. To determine the diameter of the spheroids, the phase contrast images (Zeiss Zen Blue, Carl Zeiss Microscopy Deutschland, Oberkochen, Germany) were converted to binary data using ImageJ [27 (link)], and the diameter of each spheroid was calculated.

The following software was used to collect the data in this study: Olympus Fluoview (FV31-S, 2.3.1.163) (Olympus); Zeiss ZEN Blue (v3.3.89) (Carl Zeiss Microscopy); EthoVision (XT 14) (Noldus). The following software was used to analyse the data in this study: Fiji image processing software (v1.54) (NIH); Prism (v9.2) (Graph Pad); Python (v3.9); CellRanger (v3.0) (10X Genomics); Seurat (v4.0.3); Imaris (v9.1) (Oxford Instruments).