The rats were anaesthetized intraperitoneally as described previously and transcardially perfused with heparinized saline, followed by transcardial perfusion with 4% paraformaldehyde (Sigma, China). After perfusion, the rats were decapitated, and the brain tissue was removed from the skull, embedded in OCT medium (Leica, Germany) and stored at −80°C. Coronal sections (8 μm) were obtained from the frozen brain tissue for immunohistochemical staining. The sections were covered with 5% BSA (Sigma) for 30 min at 37°C. The sections were then incubated with primary antibodies against NeuN (1:500) overnight at 4°C. The sections were washed with PBS three times and then incubated with biotinylated anti‐goat IgG (1:1000, A‐11029, Invitrogen) for 2 h at 37°C in the dark. DAPI (P36971, Invitrogen) was used to stain the nuclei. Finally, fluorescence was observed by using a fluorescence microscope (Olympus). Six sections were selected from each group, and three photos were taken of each section. Using ImageJ software (National Institutes of Health, Bethesda, MD, USA), the number of NeuN‐positive and DAPI‐positive cells in the entire photo was counted, and the NeuN‐positive cell rate was calculated and is expressed as the percentage of DAPI‐positive cells.
Oct medium
Manufactured by Leica
Sourced in Germany
OCT medium is a refractive index-matching fluid used in optical coherence tomography (OCT) imaging. It is designed to provide a transparent, uniform medium for the sample being imaged, which helps to minimize refractive index mismatch and improve image quality.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cryostat microtome, by Leica (1 mentions)
Anti tuj1, by Merck Group (1 mentions)
Donkey anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti th, by Merck Group (1 mentions)
Mouse anti nf200, by Merck Group (1 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Guinea pig anti pv, by Synaptic Systems (1 mentions)
Cryostat, by Leica (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Cm1950 clinical cryostat, by Leica (1 mentions)
7 protocols using oct medium
1
Quantifying Neuronal Cells in Rat Brain
2
Golgi Staining of Mouse Brain Cryosections
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Golgi staining were performed based on a protocol previously described [30 (link)], with modification to be compatible with cryosectioning. In short, mouse is sacrificed and perfused with saline, and whole brains were isolated and fixed in 4% PFA for 36–48 h. After fixation, brain tissues were soaked in 30 mL of 3% potassium dichromate for a week and the solution was replaced daily. Next, tissue blocks were washed in increasing concentrations (0.25, 0.50, 0.75%) of silver nitrate for 5 min each, then placed in 2% silver nitrate in the dark for another 7 days. After that, tissued were transferred into the cryoprotectant solution that contains 20% sucrose and 15% glycerol at 4 °C for 48 h, then embedded in OCT medium (Leica) and stored in − 80 °C. 100 μm-thick sections were cut by a cryostat microtome (Leica), and dehydrated in each 50%, 75%, 95%, and 100% (twice) of ethanol for 5 min respectively, then finally transferred to xylene for 5 min. Slices were mounted and then observed under a microscope (Olympus, IX73).
3
Histological Analysis of Transplanted Neural Stem Cells
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Rats were anesthetized and transcardially perfused with normal saline containing heparin and 4% paraformaldehyde (PFA; Sigma-Aldrich). The rats' brains were removed, post-fixed in 4% paraformaldehyde for 24 h, and dehydrated in 10%, 20%, and 30% sucrose at 4°C. Specimens were frozen in OCT medium (Leica, IL, USA) and then, 14-µm-thick coronal serial sections were cut and mounted on gelatin-coated slides. One brain section in each group was processed with basic hematoxylin and eosin (H&E) staining, and six sets of sections were immunohistochemically stained. Sections on the glass slides were permeabilized in PBS containing 0.5% Triton-X for 5 min at 4°C, rinsed 3 times with PBS for 5 min, and then incubated in 1% normal horse serum for 1 h at room temperature. The slides were incubated overnight in a 1∶500-diluted solution of anti-TuJ1 (Millipore Co., Billerica, MA, USA), anti-luciferase (Millipore Co.). Localization of transplanted NSCs was investigated via staining with anti-Thy1.1 and anti-luciferase antibodies. Immunohistochemistry analyses were performed using confocal laser microscopy (LSM 510; Carl Zeiss, Jena, Germany).
4
Immunofluorescence Labeling of Brain Tissue
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Brain tissue was collected and immersed in 4% paraformaldehyde for 24 hours, and then transferred to a 30% sucrose solution (0.1 M PBS, pH = 7.4) for 3 days. Brain tissue was embedded in OCT medium (Leica 020106926, Germany) in a cryo-embedding machine and stored at -80°C. Frozen brain tissues were sliced (15 µm) with a frozen slicer, blocked with 0.4% triton X-100 for 40 minutes and 5% donkey serum for 1 hour. Tissue sections were washed with PBS, and incubated with primary antibodies. For double-labeling, polyclonal rabbit anti-LC3 antibody (dilution 1 : 100; MBL) and polyclonal mouse anti-NeuN antibody (dilution 1 : 100; Millipore) were incubated simultaneously overnight at 4˚C. Sections were then washed 3 times with fresh PBS and incubated with fluorescent secondary antibodies tagged with FITC 488 (donkey anti-rabbit IgG, 1 : 200, Santa Cruz Biotechnology) or 594 (donkey anti-mouse IgG, 1 : 200, Santa Cruz Biotechnology) for 2 hours at room temperature in dark. All cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). After washing with PBS, images were collected using a laser confocal microscope (Olympus FV950).
5
Immunohistochemical Analysis of DRG and Skin
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DRG and skin (hairy and glabrous) were dissected and fixed with 4% PFA for 2 h or O/N 4°C, respectively. Tissues were cryoprotected in 30% sucrose in PBS O/N at 4°C, then embedded in OCT medium (Leica). Both DRG and skin were sectioned at a thickness of 40 μm using a cryostat (Leica). Sections were then treated with cold 50% ethanol for 30 min, followed by 2% BSA-0.3% Triton-X in PBS blocking solution for 1 h at RT, and finally incubated with primary antibodies in blocking solution O/N at 4°C. Secondary antibodies were diluted in blocking solution and incubated for 2 h at RT. Sections were mounted using DAPI-Fluoroshield™ Medium (Sigma). All images were acquired at 40× with a Leica SP2 confocal microscope and analyzed with ImageJ-Fiji (NIH, MD, 312 USA).
The following primary antibodies were used: 1:300 mouse anti-TRPV1 (Neuromics), 1:50 mouse anti-TrkA (MNAC13, 5.4 μg/μl) (105 (link)), 1:200 mouse anti-CGRP (Immunostar), 1:100 isolectin GS-B4-biotin conjugate (Invitrogen), 1:300 guinea pig anti-PV (Synaptic Systems), 1:200 rabbit anti-TH (Millipore), 1:500 mouse anti-NF200 (Sigma), 1:200 rabbit anti-PGP9.5 (Dako-Agilent), 1:300 guinea pig anti-VAChT (Synaptic System). All Alexa-conjugated secondary antibodies were used at 1:1000 concentration.
The following primary antibodies were used: 1:300 mouse anti-TRPV1 (Neuromics), 1:50 mouse anti-TrkA (MNAC13, 5.4 μg/μl) (105 (link)), 1:200 mouse anti-CGRP (Immunostar), 1:100 isolectin GS-B4-biotin conjugate (Invitrogen), 1:300 guinea pig anti-PV (Synaptic Systems), 1:200 rabbit anti-TH (Millipore), 1:500 mouse anti-NF200 (Sigma), 1:200 rabbit anti-PGP9.5 (Dako-Agilent), 1:300 guinea pig anti-VAChT (Synaptic System). All Alexa-conjugated secondary antibodies were used at 1:1000 concentration.
6
Soleus Muscle Tissue Sampling
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After the evaluation of lung mechanics, animals were sacrificed with an overdose of intraperitoneal injection of pentobarbitone (100mg/kg body weight). Immediately after sacrifice, the soleus muscles of the right hindlimb were carefully isolated and snap-frozen in liquid nitrogen and stored at -80°C for enzyme assays and ELISA assessments, while the soleus muscles of the contralateral hindlimb were embedded in O.C.T.™ medium (Leica Biosystems, Germany) under liquid nitrogen. Cross-sections of 7μm thickness were cut from the muscle mid-belly and were stored at -80°C until the subsequent histological analyses.
7
Cryopreservation and Histological Analysis of Membrane Samples
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All surgically peeled membrane samples were embedded in the optimal cutting temperature compound (OCT)-medium (Leica Biosystems, Nussloch GmbH, Germany) and stored at −20 °C initially for slow freezing. After freezing, sections of 4 µm thickness were prepared using cryostat (Leica CM1950 Clinical Cryostat-Leica Biosystems, Nussloch GmbH, Germany) for each subject. The sections were stained with hematoxylin and eosin (H&E) dyes and images were taken at 4× and 10× with an inverted microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).
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