Axioimager mi microscope

Wax-embedded samples of rheumatoid synovial tissue were sectioned and H&E stained. Antigen retrieval of deparaffinised sections was performed by microwaving in 1 mM EDTA (pH 8). Sections were exposed to anti-ANGPTL4 (H-200; Santa Cruz Biotechnology), anti-cathepsin K (clone 182-12G5, Calbiochem), anti-CD20 (clone L26; Dako), anti-CD68 (clone KP1; Dako), anti-HIF-1α (clone 54; BD Biosciences) or a serum control. For standard immunohistochemistry, staining was visualized with the VECTASTAIN Elite ABC Kit (Vector Laboratories). For immunofluorescence, secondary antibodies were DyLight 488 or 594 conjugates (Thermo Scientific). Image acquisition was performed using a Zeiss AxioImager MI microscope, AxioCam HRC camera and AxioVision software.
Osteoclasts in tissue sections were considered as large, multinucleated cells containing >3 nuclei; a widely accepted identification criterion in RA [23] (link)–[26] (link). Osteoclast identification was additionally confirmed by staining for cathepsin K; an enzyme expressed at high levels in osteoclasts [27] (link), [28] (link) and a validated osteoclast marker in RA [29] (link), [30] (link).

Spotting onto drug-free medium was performed to evaluate cidality as previously described (69 (link)). Plates were incubated at 30°C and photographed after 24 h. Propidium iodide staining was used to visualize cell death. Cells were subcultured from a saturated overnight culture to an OD₆₀₀ of 0.1 in YPD medium and grown for 18 h in the absence or presence of 50 μM CMLD010515. Cells were pelleted and resuspended in phosphate-buffered saline (PBS) with 1 μg/ml propidium iodide. Cells were imaged by differential interference contrast (DIC) microscopy and by the use of the DsRed channel on a Zeiss Axio Imager.MI microscope (Carl Zeiss) at the same exposure time.

We assessed cellular morphology of our transcription factor gene deletion mutants in the wild type (SN95) and the Hsp90 conditional expression background (MAL2p-HSP90/hsp90∆) (S1 Table). Overnight cultures at 30°C and 200 rpm were set up so that strains in the wild-type background were cultured in YPD and strains in the MAL2p-HSP90/hsp90∆ background in YPM at 30°C. To observe morphology in response to Hsp90 depletion in non-filament inducing conditions, overnight cultures were then adjusted to an OD₆₀₀ of 0.2 in YPDM medium and incubated for 6 hours at 30°C. To determine if changes in cell morphology were specific to Hsp90 depletion, transcription factor deletion mutants in the SN95 wild-type background were tested in canonical filament-inducing conditions, including 10% newborn calf serum (Invitrogen) in YPD, Lee’s Medium [32 (link)] and Spider Medium [33 (link)]. Strains grown in serum were imaged after 2 hours and those in Lee’s and Spider media after 6 hours. Images were captured using the Differential Interference Contrast setting on the Zeiss Axio Imager.MI microscope together with Axiovision software (Carl Zeiss, Inc.).

Femoral length and width (antero/posterior) were measured in fresh collected bones using a digital caliper (Ted Pella, Inc) (8–20 per age and per genotype). Percentage of tissue area and cross-sectional composition (Figure 1) were calculated based on areal measurements obtained with ImageJ Software areal measurement tool. The whole radius from newborn (n = 6–9) and P14 (n = 3–9) Bmp2 Prx1-cKO mice and control littermates were sectioned from distal to proximal. One section every 50 µm was measured. Digital images were obtained using a Zeiss AxioImager MI Microscope fitted with an AxioCam HRC digital camera and Zeiss AxioVision imaging software.

To analyse F4/80 expression digital images were acquired with an AxioImager. MI microscope (Zeiss, Germany) using an in-house Metamorph macro (Molecular Devices, USA). All images were stored in uncompressed 24-bit colour TIFF format. Images were automatically analysed using a plugin developed for Fiji, ImageJ.41.