Cm3050 s

Vastus lateralis and soleus muscle biopsies (approximately 150 mg) were taken from 21 subjects using a rongeur (4 mm diameter) under sterile conditions at baseline (BDC), Day 6 and Day 55 or 57 of head‐down tilt bed rest (HDT6 and HDT55/57). Samples were acquired after skin disinfection and local anaesthesia with lidocaine. Biopsies were mounted on cork with Tissue‐Tek O.C.T. Compound (Sakura, Torrance, CA, USA) in orientation for transverse sections, frozen in liquid nitrogen and stored at −80°C. Sections (10 μm) were cut using a cryostat (Leica CM 3050S, Leica Biosystems, Wetzlar, Germany), mounted on adhesion slides and stored at −80°C until staining. Due to issues with tissue freezing, histological analysis was completed on samples from 19 people for the vastus lateralis (control n = 6, AG n = 13) and 18 people for the soleus (control n = 6, AG n = 12).

After image acquisition, under deep anesthesia, rats were euthanized on day 28 post-MCAo,
both treated and control animals, for immunohistochemical investigations
(n = 32), and all animals from each group were submitted to staining
and measurement of infarct volume by triphenyltetrazolium chloride-2,3,5
(n = 4). Briefly, animals were transcardially perfused with a buffered
saline solution and 4% paraformaldehyde. The brains were removed and stored in
paraformaldehyde for 24 h and cryoprotected in a 40% sucrose solution for 48 h. Coronal
sections were cut to 40 μm thickness using a cryostat (Leica CM3050 S, Leica Biosystems)
and stained using standard procedures for hematoxylin-eosin, periodic acid–Schiff (PAS),
and Masson Trichrome staining and the proliferation marker Ki67. Coronal sections also
were cut to 1 mm thickness using a cryostat (Leica) and stained using triphenyltetrazolium
chloride-2,3,5 (TTC) (Sigma-Aldrich) at 37°C for 30 min to determine the viability of
brain tissue. The infarcted areas were pale, while the normal brain tissue was stained
red. The slices were photographed and analyzed in the image J software (NIH Image
Software, Bethesda, MD, USA) to determine the infarct volume. The extent of tissue damage
was calculated as a percentage of the infarct volume^{30 (link)}.

Islet graft-bearing kidneys, pancreases, and intestines fixed in 4% paraformaldehyde were embedded in optimal cutting temperature compound and frozen on dry ice. 6–10μm sections were made on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Immunofluorescent staining was performed using standard methods. Briefly, sections were blocked in 5% BSA for 1hr then incubated with primary antibodies overnight at 4°C. Sections are washed 3 × 5 min before incubation with secondary antibodies for 2 hours at room temperature and washed 3 × 5 min again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher). Post-processing and color channel merging was performed in Fiji (http://fiji.sc/) (Schindelin et al., 2012 (link)). Primary antibodies (1:100–200): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (a0564) from Dako (Carpinteria, CA). Secondary antibodies (1:200–500): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO), Alexa Fluor-594 and Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.