Axio imager z2

Paraffin embedded human mesothelioma tumour tissue sections (5 μm), prepared from tumours excised from the four xenograft treatment groups at Day 39 (Saline, ^AmatuxEDV, ^Non-targetedEDV_Dox, ^AmatuxEDV_Dox) were dewaxed in xylene, rehydrated in graded ethanol solutions. Tissue samples were then stained for 7 min in Mayers Haematoxylin and 20 sec in 1% Eosin. Samples were then dehydrated through graded ethanol solutions and cleared with xylene. Finally, slides were coverslipped using DPX solution (Sigma). Samples were visualised using an upright bright-field slide scanner (Zeiss Axio Imager Z2). The percentage of necrotic areas was estimated by blind measuring of multiple scattered necrotic regions (absence of nuclear staining) using 20x magnification. The percentage was estimated from three different mice representing each treatment group. Images were captured using a Zeiss Axio Imager Z2 slide scanner and analysed using ImageJ software v1.50i (National Institutes of Health, Bethesda, MD). The mean values for the percentage of necrotic regions for all treatments were analysed for variance using one-way ANOVA with Tukey post-hoc test. Statistical significance was determined as p ≤ 0.05 [47 (link)].

Adrenal medullae (randomly selected five sections per treatment group) were im-aged (Zeiss Axio Imager Z2; 20×) and analyzed in Fiji ImageJ software. The thresholded Fos-positive area, measured in pixels, was normalized to the PNMT-positive area and ex-pressed as a percentage of PNMT-positive area. Fos-positive staining outside of the PNMT-immunoreactive area was excluded. Four 40 µm brainstem sections containing the C1 catecholaminergic nucleus (bregma level −12.48 to −12.12) per animal were used to count the number of TH immunoreactive cells (TH⁺), Fos immunoreactive cells (Fos⁺), and double-labeled (TH⁺/Fos⁺) cells bilaterally within the region. A rectangular region of interest (ROI, 1680 × 810 µm) was superimposed on the images taken with Zeiss Axio Imager Z2, 10× objective, and cells were counted manually within the ROI by an operator blinded to the experimental conditions.

Retinal organoids were collected at days 35, 90, and 150, washed with PBS for 10 minutes and then fixed in 4% paraformaldehyde for 15 minutes. Retinal organoids were washed again with PBS before being placed in 30% sucrose overnight. Retinal organoids were placed into cryogenic molds and immersed in optimum cutting temperature (OCT). About 10 μM slices were sectioned on a Leica cryostat (CM1850). Cut sections were blocked in 0.3% Triton‐X‐100 (Sigma) and 10% NGS (Thermo Scientific) in PBS for 1 hour. This solution was removed and replaced with antibody diluent (0.3% Triton‐X‐100, 1% BSA in PBS) with applicable antibody dilution (Supporting Information Table S2) at 4°C overnight. Sections were washed three times in PBS followed by incubation with secondary antibodies for 2 hours at room temperature. Sections were washed again as stated previously and mounted in Vecta‐shield (Vector Labs, Burlingame, CA) with Hoechst 33342 (1:1000, Thermo Scientific) or DAPI (Sysmex, 1:1000). Images were captured using Nikon A1R confocal (resonant, invert; Nikon, Japan) and Zeiss Axio ImagerZ2 equipped with an Apotome2 (Zeiss, Germany).

OCT-12 μm slices of retinal organoids collected at day 150 of differentiation were stained with Hematoxylin and Eosin. Air-dried sections were incubated for 7 minutes at room temperature with Hematoxylin (LAMB/230-D), uniformly covering the tissue on the slide. Slides were washed in Milli-Q water, followed by 2-minute incubation at room temperature with Bluing Buffer (DAKO, CS702), repeated washes and final 1 minute incubation at RT with Eosin Mix (Eosin 1% AQUEOUS, LAMB/100-D) diluted 1:10 in tris-acetic buffer; 0.45 M, pH 6.0). The mix was then drained, slides washed with Milli-Q water, air-dried, and mounted with 85% glycerol (Merck Z0566194921). Bright-field images were taken using Zeiss Axio ImagerZ2 (Zeiss, Germany). The percentage of rosette-like structures was calculated by dividing the number of rosettes by number of cells in the section × 100.

To assess cell proliferation, frozen bone sections were rehydrated in PBS, and paraffin ileum sections were deparaffinized and rehydrated in graded alcohol. A 1% trypsin in PBS solution was placed on the tissue for 10 min at 37°C. Slides were rinsed in PBS followed by 4 N HCl for 30 min at room temperature. Slides were rinsed in PBS then blocked in mouse‐on‐mouse blocking reagent (Vector Labs, Burlingame, CA, USA) for 10 min. A biotinylated monoclonal BrdU antibody (B35138, diluted 1:50 in PBS; Thermo Fisher Scientific, Waltham, MA, USA) was placed on the tissue and incubated overnight at 4°C. Frozen sections were brought to room temperature, rinsed in 1X PBS, and incubated with a Alexa Fluor 488 Streptavidin conjugate (S11223, diluted 1:100 in PBS; Thermo Fisher Scientific) for 1.5 hours. Sections were then rinsed and cover‐slipped with Prolong Diamond Antifade with DAPI (P36966; Thermo Fisher Scientific). Following antibody treatment, paraffin ileum sections proceeded with a HRP‐streptavidin and DAB substrate reaction and were counterstained with hematoxylin. For paraffin histology, bright‐field BrdU images were obtained using the Zeiss Axio Imager Z2 microscope and a 20× objective. For frozen histology, fluorescent images were obtained along the periosteal compressive surface on the Zeiss Axio Imager Z2 with the GFP filter used to detect BrdU Alexa 488.

Super-resolution STED and related CLSM images were acquired at the same position using the STEDYCON compact line nanoscope (Abberior Instruments GmbH, Göttingen, Germany) mounted on an Zeiss Axio Imager Z2 (Carl Zeiss) microscope. The STEDYCON nanoscope unit is equipped with a 405 nm continuous wavelength laser, a 640 nm pulsed excitation laser and a 775 nm pulsed STED laser; oil immersion objective (Plan-Apochromat 100×/N.A. 1.46 Oil DIC M27) and avalanche diode detectors (APDs). DAPI and DyLight633 fluorescence were excited using the 405 nm and the 640 nm lasers, respectively. The pinhole size was 64 μm. The fluorescence was split and detected by distinct APDs (DAPI: 420–475 nm, DyLight633: 650–700 nm). The STED laser power was set to 98% (maximum power) to acquire images with the highest spatial resolution possible.