Neutral buffered formalin

AC tissue was separated into two and either fixed in 10% v/v neutral buffered formalin (Leica) and processed to paraffin wax, or processed for cell extraction. Following paraffin wax embedding, 4μm sections were histologically graded based on the Mankin [23 (link)] grading system with the addition of abnormal features and cartilage thickness also assessed. Cells were extracted using trypsin and collagenase as previously published [24 (link)], and cells were used for direct RNA extraction using TRIZOL reagent and cDNA synthesised.

Kidneys were isolated from resting mice after PBS perfusion and fixed O/N in 20 mL of 10% Neutral Buffered Formalin (Leica). Fixed kidneys were paraffin-embedded using a Shandon Citadel 1000 tissue processor (Thermo Fisher Scientific) and stored at room temperature (RT) until used. The day prior to CCR1 mRNA analysis, kidneys were wax-embedded and sliced into 6 μm sections onto SuperFrost Plus adhesion slides (Thermo Fisher Scientific). Slides were air-dried O/N at RT and, the following day, CCR1 mRNA was detected using an RNAscope target probe specific for the gene with the RNAscope 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics). Manufacturer’s instructions were followed, with minor modifications. Specifically, kidney sections were incubated in the RNAscope Target Retrieval Reagent for 18 min and were treated with the RNAscope Protease Plus Reagent for 35 min. Images were acquired on an Evos FL Auto 2 microscope (Thermo Fisher Scientific).

Bovine serum albumin (BSA, 7906, Sigma-Aldrich, St. Louis, MO, USA), DAPI (D1306, ThermoFisher Scientific, Santa Clara, CA, USA), 16% Formaldehyde (w/v), Methanol-free (Cat #28908, Pierce™ ThermoFisher Scientific, Santa Clara, CA, USA), 10% Neutral Buffered Formalin (3800598, Leica), Triton-X 100 (T8787, Sigma-Aldrich, St. Louis, MO, USA), Tween-20 (170-6531, Bio-Rad), and U0126 (#9903, Cell Signaling Technology, Danvers, MA, USA).

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).