Bz x800 series microscope

Murine adherent cells were seeded onto 96-well hydroxyapatite-coated plates (OsteoAssay-Corning) using osteoclastogenic media, as described in our osteoclast differentiation protocol. After 4 days, the cells were removed, and the extent of bone resorption was quantified by measuring the size and number of resorption pits formed by osteoclasts on the bone substrate. Image analysis software (ImageJ) was employed to calculate the pit area and resorption activity after acquiring images in a Keyence BZ-X800 Series microscope using a 10× objective lens. The investigator was blinded during all analyses.

In situ hybridization was performed on 20 μm cryotome coronal brain sections − 1.22 mm and − 1.46 mm caudal from Bregma using RNAscope^® Probe- Mm-Pmch-C2.
(Cat No. 478721-C2). Selected sections were mounted on superfrost slides and processed using reported method by manufacturer (Advanced Cell Diagnostics, Newark, CA, ACD, protocol 320,293-USM). Epifluorescent images 20X tile and Z-stack with 1 μm thick optical section were acquired with a Keyence BZ-X800 series microscope, and run by the BZ-X800 analyser software. Pmch-positive neurons containing DAPI positive nucleus were manually counted, on maximal intensity projection (MIP) images. Experimenters were blinded to the genotypes performed the neuronal counts.

FFPE tissue sections (5 μm thick) for six cases of LSCC and matched histologically normal lung tissues from two of these cases and four additional matched histologically normal lung tissues were deparaffinized and rehydrated in ethanol and water. Endogenous peroxidase was inactivated with 3% hydrogen peroxide for 10 min at room temperature. After antigen retrieval with citric acid buffer (10 min, 95^◦C), sections were blocked with background sniper (Biocare, BS966) then incubated for 1 h at room temperature with a knock-out validated recombinant rabbit monoclonal anti-SERPINH1 (Hsp47) antibody (Abcam, #ab109117) diluted at 1/50 or 1/150 followed by an incubation for 1 h at room temperature with a secondary goat Alexa Fluor™ 488 antibody (ThermoFisher Scientific, #A11029) diluted at 1/1000. Tissue sections were counterstained with DAPI, mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, H-1400) and coverslipped. Sections were then imaged, specifically acquiring five images per each specimen, at a 20× magnification using a Keyence BZ-X800 series microscope. Images were processed with the ImageJ software, and Hsp47 abundance was quantified based on fluorescein isothiocyanate (FITC) staining using the QuPath (version 0.3.2) software. Plots and statistical analysis (Welch’s test) were processed with the Prism (version 9.3.1) software.