Neutral buffered formalin

Lung samples were fixed overnight with 10% Neutral buffered formalin (Leica Biosystems Inc., Buffalo Grove, IL, United States), dehydrated using 30% sucrose overnight then embedded in optimal cutting temperature (OCT) compound, snap-frozen by liquid nitrogen, and sectioned at 7 μM. Heat mediated antigen retrieval was performed for 20 min in a steamer using Citrate Buffer (Abcam, Cambridge, MA, United States). Slides were incubated with mouse monoclonal anti-Arl13b antibody (75–287, clone N295B/66, UC Davis/NIH NeuroMab Facility, Davis, CA, United States) at 4°C overnight and then incubated with Cy3-rabbit anti-mouse antibody (Jackson ImmunoResearch Labs, West Grove, PA, United States) for 1 h at room temperature (RT), the following day. Nuclear staining and mounting were performed using Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, United States). The percent ciliation was expressed as percentage of Arl13b⁺ cells per section were quantified using ImageJ software from at least six sections that were 100 μm apart for each mouse offspring. This measurement was expressed as percent ciliation.

A total of 14 Sprague Dawley rats (female, 10–14 weeks old) were used in this study with two for cervical imaging in vivo and 12 for vaginal imaging ex vivo. The estrous stage was evaluated by the features of exfoliated vaginal cells prior to μOCT image acquisition. Specifically, the rats were housed under a standard 12:12 light–dark cycle, and vaginal cell samples were collected at 10:00 a.m. every day using the pipette smear technique.56 Following sample collection and smearing, the air‐dried cells were stained with 0.1% crystal violet solution (Sigma‐Aldrich) for 1 min, washed twice in ddH₂O to remove excess stain and examined under a light microscope (Olympus BX53).
For cervical study in vivo, rats at the proestrus were anesthetized and we performed μOCT imaging after surgical exposure of the squamocolumnar junction. For vaginal study ex vivo, rat was sacrificed and μOCT images were acquired from the luminal side of the upper third of the vagina within 30 mins after tissue harvest. Following image acquisition, regions of interest were fixed with 10% neutral‐buffered formalin (Leica Biosystems) and 5 μm‐thick sections were stained with H&E for histological analysis. These studies were approved by IACUC of NTU (ARF‐SBS/NIE‐A0312).

Homozygous mice (7M and 7F) from each of the alleles (Slc25a21^{tm1a(KOMP)Wtsi}, Slc25a21^{tm1b(KOMP)Wtsi}, Slc25a21^{tm1c(KOMP)Wtsi} and Slc25a21^{tm1d(KOMP)Wtsi}), as well as age, sex and genetic background matched controls, were analysed using the standard Sanger Institute Mouse Genetics Project phenotyping screen [2] (link). Assays of particular relevance are described in File S1, whilst those not part of the standard phenotyping screen are described below.
At necropsy, skulls were collected and the teeth (both upper and lower jaw) and palate were reviewed and imaged (MZ16A dissecting microscope, Leica, Wetzlar, Germany; DFC490 digital camera, Canon Powershot G5, Japan). A range of 42 additional tissues and organs from homozygous Slc25a21^{tm1a(KOMP)Wtsi} mice (2M and 2F), and age, sex and genetic background matched controls, were fixed in 10% neutral buffered formalin (Leica Biosystems, Peterborough, UK) for 15–20 hrs and processed to wax. 5 µm sections were stained with haematoxylin and eosin then reviewed by an experienced pathologist.
Micro-CT (computed tomography) images of skulls from Slc25a21^{tm1a(KOMP)Wtsi} mice (wt, n = 2; hom, n = 2) were collected at 18 µm resolution (SkyScan 1176, SkyScan, Kontich, Belgium).