Mandibles and maxillae were carefully dissected, briefly washed in PBS, and fixed in 4% paraformaldehyde (pH 7.4) for 5 hours at 4°C. Fixed tissue was then decalcified in 10% EDTA (pH 7.4) for 10 days at 4°C. Samples were then processed either for cryosectioning or paraffin processing. Briefly, for cryosectioning, samples were incubated in 30% sucrose (Sigma‐Aldrich, St. Louis, MO, USA; 16104) overnight at 4°C, washed twice in Tissue‐Tek OCT Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands; 4583) and embedded in fresh OCT. Samples used to obtain detail images were then sectioned to a thickness of 14 μm on cryostat Leica CM1860 (Leica Biosystems, Wetzlar, Germany). To obtain overview images of the mandible and maxilla of DSPPCerulean/DMP1Cherry, samples were sectioned to a thickness of 40 μm using Kawamoto's tape (SECTION‐LAB Co. Ltd., Hiroshima, Japan) on cryostat Leica CM1860 (Leica Biosystems). Samples embedded in paraffin were sectioned on a microtome (Leica SM2000R) to a thickness of 2 μm and processed as described further.
Cm1860
Manufactured by Leica
Sourced in Germany, United States, Japan, China, United Kingdom, Canada
The Leica CM1860 is a cryostat, a device used for the preparation of frozen tissue samples for microscopic examination. It features a built-in microtome for sectioning the samples and a temperature-controlled chamber for maintaining the desired freezing conditions.
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Lab products found in correlation
Tissue tek oct compound, by Sakura Finetek (7 mentions)
Frozen section compound, by Leica (6 mentions)
Benzocaine solution, by Merck Group (6 mentions)
Silane coated glass slides, by Muto Pure Chemicals (5 mentions)
Anti gaba, by Merck Group (5 mentions)
Image pro plus 6, by Media Cybernetics (4 mentions)
Lsm 710, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Fluoromount g, by Southern Biotech (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Lsm 800, by Zeiss (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Rm2235, by Leica (4 mentions)
Tissue tek, by Sakura Finetek (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Axiovision 3.0.6 sp4, by Zeiss (3 mentions)
Tcs sp8 confocal microscope, by Leica (3 mentions)
Abcam5683 rabbit polyclonal, by Abcam (3 mentions)
Tissue tek oct, by Sakura Finetek (3 mentions)
Anti c fos, by Santa Cruz Biotechnology (3 mentions)
290 protocols using cm1860
1
Preparation of Craniofacial Tissues for Microscopy
2
Immunohistochemical Analysis of c-Fos in LPBN
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Brains samples were fixed with 4% paraformaldehyde for 24 h and were transferred into 30% sucrose at 4°C for 12 h. Samples were then cryo-sectioned into 15 μm sections by microtome (CM1860; Leica) and dried at 60°C in an incubator (DHG-9101; SANFA, Yangzhou, China) for 4 h. Sections were then hydrated in 0.01 M PBS for 15 min, antigen retrieval was performed following manufacturer’s instructions (P0083, Beyotime, Beijing, China). Sections were further washed with 0.01 M PBS for three times, blocked with 0.1% FBS for 1 h at 37°C, and then incubated with a c-fos primary antibody (rabbit antimouse, ab190289, 1:1000; Abcam) for 2 h at 37°C and HRP (horse radish peroxidase) secondary antibody (PV-6001, 1:200; Zsbio, Tianjin, China) for 20 min at 37°C. Between incubation by antibody and after incubation by secondary antibody, sections were washed as described previously. A DAB kit (ZLI-9018; Zsbio) was used to develop the staining. Morphology was assessed using a light microscope (CX31; OLYMPUS). According to the mouse brain in stereotaxic coordinates (36 ), LPBN is located in the dorsolateral pons that surrounds the superior cerebellar peduncle (scp), which we considered as the neuroanatomical landmark to delimit LPBN in this work. By using ImagePro Plus 6 (Media Cybernetics, Inc. USA), the number of c-fos immunopositive neurons within LPBN were counted.
3
Immunofluorescent Staining of Spinal Cord
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Coronal and sagittal sections of spinal cord samples with 12 μm were obtained using a cryostat (Leica, CM1860). The slides were incubated overnight with primary antibodies (Table 1 ) in a solution containing 5% normal donkey serum (Sigma, D9663) and 0.5% Triton X-100 diluted in PB 0.1 M at room temperature. After serial washes with PB 0.1 M, sections were incubated with 1:500 Alexa488 fluorescent secondary antibody diluted in 0.5% Triton X-100 on PB 0.1 M for two hours at room temperature. The slices were counterstained with 4′-6-diamino-2-phenylindole (DAPI). For double IF, the same protocol was performed using Alexa 546 secondary antibody.
4
Tissue Preparation for Western Blotting and Immunofluorescence
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Human tissues were immediately frozen in liquid nitrogen and stored at −80 °C for western blotting. Animal tissues were immediately divided into 1 portion that was immediately frozen in liquid nitrogen and then stored at −80 °C for western blotting and a second portion that was fixed in 4% phosphate-buffered formalin for 48 h. This portion was frozen and cut into 15-μm sections with a cryostat microtome (Leica CM1860, Germany) for double immunofluorescence labelling and stored at −20 °C.
5
Immunophenotyping of Tumor-Associated Macrophages
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Tumor chunks (n = 30) were fixed in 4% paraformaldehyde overnight, instilled in
30% sucrose, and embedded in optimal cutting temperature (OCT) medium, snap
frozen, and sectioned at 5 µm (Leica CM 1860). Tissue sections were
permeabilized and blocked prior to antigen retrieval with citrate buffer.
Sections were incubated with CD80 (1:500, #8679; ProSci) or CD163 (1:500, #163M;
Cell Marque) primary antibodies at 4°C overnight, Alexa Fluor 594–conjugated
secondary antibody (Life Technologies) for 2 hours at room temperature, and
4′6-diamidino-2-phenylindole (DAPI) nuclear stain for 10 minutes. Antifade
mounting medium was added before cover-slipping. Confocal images were obtained
for 3 different areas (Zeiss LSM 700 Microscope, 40× oil immersion lens).
Serum-treated sections were used as negative controls. After the minimum and
maximum threshold values were set to eliminate nonspecific staining and artifact
on ImageJ software (National Institutes of Health), expression levels for CD80
and CD163 were determined as percent area per region of interest on high-powered
field. Median values were compared between samples.
30% sucrose, and embedded in optimal cutting temperature (OCT) medium, snap
frozen, and sectioned at 5 µm (Leica CM 1860). Tissue sections were
permeabilized and blocked prior to antigen retrieval with citrate buffer.
Sections were incubated with CD80 (1:500, #8679; ProSci) or CD163 (1:500, #163M;
Cell Marque) primary antibodies at 4°C overnight, Alexa Fluor 594–conjugated
secondary antibody (Life Technologies) for 2 hours at room temperature, and
4′6-diamidino-2-phenylindole (DAPI) nuclear stain for 10 minutes. Antifade
mounting medium was added before cover-slipping. Confocal images were obtained
for 3 different areas (Zeiss LSM 700 Microscope, 40× oil immersion lens).
Serum-treated sections were used as negative controls. After the minimum and
maximum threshold values were set to eliminate nonspecific staining and artifact
on ImageJ software (National Institutes of Health), expression levels for CD80
and CD163 were determined as percent area per region of interest on high-powered
field. Median values were compared between samples.
6
Immunofluorescence Staining of Brain Sections
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Animals were anesthetized with sodium pentobarbital and perfused transcardially with ice-cold saline followed by 4% paraformaldehyde. Then, the brains were isolated and perfused in 30% sucrose in PBS. Coronal brain sections (25–30 μm) were cut by microtome (Leica, CM1860) and stored at 4°C. Brain slices were washed in PBS 3 times, and then permeabilization and blocking were performed in PBS containing 0.3% Triton X-100, 1% BSA, and 10% goat serum at room temperature for 1 hour. Slices were incubated with the primary antibodies overnight at 4°C and were washed 3 times the next day with PBS containing 0.1% Triton X-100, followed by incubation with fluorescence-conjugated secondary antibodies (Abcam, 1:1000) for 1 hour at 37°C. Finally, after counterstaining with DAPI, images were captured with an Olympus confocal microscope. The following primary antibodies were used in the immunofluorescence assay: anti-EphA4 (Abcam, ab5396, 1:50 or Santa Cruz Biotechnology, sc-365503, 1:50); anti-GFAP (Abcam, ab68428, 1:500); anti-Iba1 (Wako, 019-19741, 1:1000); anti-Vglut1 (Servicebio, GB11821, 1:500); anti-GAD65 and anti-GAD67 (Santa Cruz Biotechnology, sc-365180, 1:100); and anti-MBP (Servicebio, GB12226, 1:800).
7
Immunohistochemistry Tissue Preparation Protocol
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Tissue preparation, sectioning and staining for immunohistochemistry were performed as previously described.18 (link) In brief, animals were deeply anesthetized with isoflurane and transcardially perfused with chilled 4% paraformaldehyde/PBS solution. For immunofluorescence, brains were dissected, post-fixed in 4% paraformaldehyde/PBS overnight at 4°C, incubated overnight in sucrose solution and embedded in Tissue-Tech O.C.T. compound (45,833, Sakura Finetech). Thirty μm-thick sections were obtained with a cryostat (CM1860, Leica) and subsequently processed for immunofluorescence. Primary antibodies used for the immunostaining were rabbit anti-cFos (1:2,000, AB2572236, EnCor Biotechnology), goat anti-mCherry (1:15,000, AB0040-200, SICGEN), rat anti-GFP (1:5,000, 04,404-84, Nacalai Tesque), goat anti-Iba1 (1:100, 011–27991, WAKO) and rabbit anti-GFAP (1:2000, HPA056030, ATLAS ANTIBODIES). Secondary antibodies were Alexa Fluor 488 donkey anti-rat, 488 donkey anti-rabbit, 594 donkey anti-rabbit and 594 donkey anti-goat (1:1,000, Invitrogen). For nuclei staining, sections were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) (Cellstain- DAPI solution, D523, 1:5000, Dojindo) during the first washing step with PBS after the secondary antibody reaction. Brain sections were observed by using a laser confocal microscope (TCS SP8, Leica Biosystems).
8
Neuroanatomical Analysis of Rat mPFC
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During the following processes, all the experiments and data analyses were performed blind to treatment conditions. After the behavioral testing, under deep anesthesia with an i.p. injection of 1% sodium pentobarbital (4 mL/kg body weight), five rats from each experimental group were randomly selected and perfused transcardially with 4% paraformaldehyde. Brains were removed and split into two hemispheres by a midsagittal section. The right or left hemisphere from each rat was sampled at random and coronally sectioned into 60 μm sections on a cryostat microtome (CM1860, Leica). The sections were kept in anatomical series. From the sections containing the mPFC, every 6th section was sampled in a systematic-random manner with 15 sections per series on average. In the end, six sets of sampled sections were acquired.
9
Cryosectioning and DAPI Staining
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Decellularized samples were frozen in a −150 °C freezer, embedded in an optimal cutting temperature solution (O.C.T. Sakura Fineetek 4583, Torrance, CA, USA) and sectioned in a cryostat (LEICA, CM1860) at −30 °C. Microsections of 10 μm were transferred to frosted-edged glass slides, thawed at room temperature for 15 min, and stained with 4′,6′-diamino-2-phenyl-indole (DAPI) solution [1:10,000] in 1× phosphate-buffered saline (PBS) for 10 min in the dark. Then, the slides were washed with distilled water, observed, and photographed under a fluorescence microscope (NIKON Eclipse 80i) to verify the presence or absence of cellular nuclei.
10
Spinal Cord Tissue Extraction and Cryosectioning
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Six weeks after SCI, mice were deeply anesthetized with isoflurane, and then perfused with 50 ml 0.9% saline solution followed by 50 ml 4% formaldehyde (dissolved from paraformaldehyde in 0.1 M PBS, pH 7.4) through the left cardiac ventricle. The vertebral column was dissected out and post-fixed in 4% formaldehyde solution for 1 week. The spinal cord was peeled out, immersed in 20% sucrose solution in PBS for 48 h at room temperature (RT) and then embedded in Tissue-Tek (4583, Sakura Finetek, Torrance, USA). Serial longitudinal coronal sections of the mouse spinal cord at a thickness of 8 μm were obtained using a cryostat (CM1860, Leica, Nussloch, Germany), and sampling of sections was always performed in a standard sequence so that sections were 80 µm apart and placed together on one glass slide for standard comparison.
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