Rm 9106

Paraffin-embedded transverse sections (4 μm) from formalin-fixed uterine or vagina specimens were stained as previously described [16 (link)] with anti–Ki-67 antigen (RM-9106; Thermo-scientific). Sections were examined after numerisation using NanoZoomer Digital Pathology®. To examine the proliferative effect of each treatment, the ratio of Ki-67–positive epithelial/total cells number on the entire luminal epithelium from each uterine or vaginal section was evaluated. The luminal epithelial height (LEH) was measured from the basal membrane to the apical surface as previously described for the uterus [16 (link),17 (link)]. The values are the mean of 10 measurements in each transverse uterus or vaginal section.

Slides were washed in PBS, followed by incubation in 3% H₂O₂ in methanol for 10 min. Tissues were subjected to antigen retrieval with 10 mM sodium citrate (pH 6.0) at 95⁰ C for 15 min, and blocked with 5% NGS for 1 hr at room temperature. Tissues were incubated overnight with rabbit anti-Ki67 primary antibody (1:200 dilution; Cat# RM-9106, ThermoFisher Scientific, Waltham, MA, USA; RRID: AB_2341197) at 4⁰ C. The rest of the processing with Alexa 546 and Sytox green was the same as above.

Eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at room temperature. The intact retinas were collected, blocked, and permeabilized in PBS containing 10% goat serum, 3% BSA, 1% Triton X-100, and 0.2% Tween 20 for 1 hour. Samples were then incubated with primary antibodies against rabbit PKM2 (1:100, 4053, Cell Signaling Technology), rabbit Ki67 (1:200, RM-9106, Thermo Scientific), rabbit Fabp5 (1:200, 2.5 μg/mL, RD181060100, BioVendo), rabbit Igf1 (10 μg/mL, AF791, R&D Systems), rat F4/80 (1:100, ab6640, Abcam), rabbit IBa1 (1:400, 019-19741, Sakura Finetek), goat IBa1 (1:400, ab48004, Abcam), rat CD11b (5 μg/mL, 561114, BD Biosciences), and Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–labeled Griffonia simplicifolia isolectin B4 (10 μg/mL; catalogs 121411, 121413, and I32450, respectively; Invitrogen) overnight at 4°C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Invitrogen) for 1 hour, and then counterstained with DAPI (Invitrogen). Retinas were flat mounted on microscope slides in a mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy (Zeiss 780; Carl Zeiss). For all immunofluorescence experiments, parallel groups of tissues were stained with IgG isotype and secondary antibody as negative controls.

Mouse kidneys were fixed in 10% neutral buffered formalin and paraffin embedded. Sections (4 μm) were cut and stained for Ki67 (1:100 Thermo RM-9106), Cytokeratin (Cytokeratin, Pan Ab-1, Mouse Monoclonal Antibody 1:100, Thermo, MS-34) and p21 (1:500 Santa Cruz, sc471).