Tissue tek optimal cutting temperature oct

For hippocampal sections: brains were harvested from adult male Amigo2-EGFP transgenic mice (25–35 g, 6–8 weeks) born from separate litters. The Amigo2-EGFP line was acquired from GENSAT (founder line LW244, RRID:MMRRC_033018-UCD) and bred for at least 10 generations onto C57bl6/n background. Mice were deeply anesthetized with Fatal Plus (50 mg/kg) before decapitation and swift removal of the brain (<2 min). Brains were bisected in the sagittal plane and individually flash frozen in 22 × 22 mm disposable cryomold (Polysciences, Inc., Warminster, PA, USA) filled with Tissue-Tek Optimal Cutting Temperature (OCT, Sakura Finetek) compound and then submerged in isopentane cooled to −20°C in a dry ice and ethanol slurry. For midbrain sections: adult C57bI6 mice were treated with a single systemic injection of LPS (5 mg/kg) or saline at 2 months old and a body weight of roughly 22–24 g for an unrelated study. After 10 months, brains were removed and immediately embedded in 4557 standard cryomold (Tissue-Tek^®, Sakura) with OCT on dry ice. For liver sections: liver tissues were harvested from male C57bI6 mice (15–20 g, 6 weeks) and embedded as above for midbrain sections. Frozen tissue blocks were stored at −80°C until processing.

When mice showed the first signs of paralysis in hind limbs or at 120 days, euthanasia was proceeded with the guidelines of UC Berkeley’s OLAC administration. For histological comparison, some ALS mice were euthanized at 120 days. Spinal cords and muscles (gastrocnemius and tibialis anterior) were isolated. Then, spinal cords were fixed with 4% paraformaldehyde overnight at 4 °C and muscle tissues were fixed for 30 min at room temperature. Then, all tissues were transferred to 30% sucrose solution overnight and embedded in tissue-tek optimal cutting temperature (OCT, Sakura Finetek, The Netherlands) and snap frozen in isopentane cooled to − 70 °C with dry ice. Then, all samples were stored at − 80 °C before use.
For histological analysis, all OCT-embedded tissues were cryosectioned by a Cryostar NX50 (ThermoFisher). Muscle tissues were sectioned to 20-µm thickness while spinal cords were cut at 10 µm.

At the end of perfusion, atria and vasa were discarded and ventricles were fixed for 3 h r.t. with 4 % PAF with gentle stirring. Tissues were then washed with PBS, submerged in a solution of 30 % sucrose in PBS, allowed precipitating overnight at 4 °C and then incubated 30 min r.t. in a solution 1:1 of 30 % sucrose in PBS and TissueTek^® Optimal Cutting Temperature (O.C.T.™, Sakura FineTek, USA). Finally, tissues were embedded in O.C.T. and stored at −80 °C. 10 µm thick transverse slices were obtained starting from the apex with a CM 1900 cryostat (Leica Microsystem S.r.l.), placed onto Superfrost™ glass slides (Thermo Scientific, USA) and conserved at −20 °C. Sections used for ventricle reconstructions and 40× analyses were rinsed with PBS and, after nuclear staining for 15 min r.t. with Hoechst-33342 5 µg mL⁻¹, they were mounted with Mowiol and conserved at 4 °C. Sections for ultrastructural analysis were processed for immunofluorescence. Briefly, they were rinsed in PBS, permeabilized for 20 min r.t. with 0.5 % Triton X-100 and blocked for 1 h r.t. with 6 % BSA and 2.5 % NGS in PBS. The primary antibody mouse anti-sarcomeric α-actinin 1:600 in PBS was incubated overnight 4 °C, whereas the secondary antibody anti-mouse Alexa Fluor 488 1:1000 in PBS was incubated for 1 h r.t. Finally, samples were mounted with Mowiol and conserved at 4 °C.

Mice were sacrificed per the guidelines of UC Berkeley’s and Buck Institute’s OLAC administration. Blood was collected by terminal cardiac puncture and was allowed to clot completely at room temperature for at least 30 min. Clotted blood samples were centrifuged at a speed of 5000g for 5 min in order to obtain serum. Post-mortem isolation of brain was performed. Tissues were embedded in Tissue-Tek optimal cutting temperature (OCT, Sakura Finetek, The Netherlands) and snap frozen in isopentane cooled to − 70 °C with dry ice.

Mice were sacrificed per the guidelines of UC Berkeley’s OLAC administration. Blood was collected by terminal cardiac puncture and was allowed to clot at room temperature over 30 minutes. Blood serum for proteomics analysis was obtained by centrifuging clotted blood samples at a speed of 5,000 g for 5 minutes. Postmortem isolation of muscle, liver, and brain was performed. Tissues were embedded in tissue-tek optimal cutting temperature (OCT, Sakura Finetek, The Netherlands) and snap frozen in isopentane cooled to -70°C with dry ice.

Immunofluorescence microscopy was done on pancreatic tissue cryosections embedded in Tissue-Tek® Optimal Cutting Temperature (OCT) (Sakura Finetek USA, Inc., Torrence, CA), AR42J cells or acini plated on plain glass coverslips. These were fixed with 2% paraformaldehyde and processed as described previously^{15 (link),30 (link)}. After blocking with 5% normal goat serum, tissue cryosections were incubated with primary antibodies (1:50 for cryosections and 1:200 for cells) for 1 h at room temperature, washed, and incubated with secondary antibodies (Alexa 488- or Alexa 594-conjugated, diluted 1:500) Cy5-conjugated phalloidin (100 nM) (ThermoFisher, Waltham, MA) with or without DRAQ5 (1:5000) for 30 min. After washing and mounting in Fluoromount G (Sigma Aldrich) on SuperFrost slides (ThermoFisher), confocal imaging (1 µm thick) was done using a Zeiss LSM800 confocal microscope (Thornwood, NJ). Images were collected with a Zeiss C-Apochromat 63x/1.2 NA water immersion objective.