The murine brains were fixed through transcranial perfusion with 4% ice-cold paraformaldehyde, as previously reported [47 (link)], for 72 h in 4% paraformaldehyde and then, with 20% sucrose for an additional 72 h at 4 °C. The brains were mounted using the optimal cutting temperature (OCT) compound (Tissue-Teks O.C.T. Compound Medium, Sakura Finetek USA, Inc., Torrance, CA, USA), frozen in liquid nitrogen, and sectioned (14 µm) in the coronal plane using a CM 3050C cryostat (Leica, Wetzlar, Germany). For immunofluorescence analysis, the slides were washed twice with 1× PBS, incubated with proteinase K solution and blocked with normal goat/rabbit serum (5% normal goat/rabbit serum, 0.3% Triton X-100, and PBS). The brain slices were incubated with primary antibodies (1:100 in PBS) overnight at 4 °C and then with fluorescence-conjugated (FITC and TRITC from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling Technology ((Danvers, MA, USA), and Abcam, secondary antibodies (in PBS) at room temperature. The nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole). The slides were mounted with glass coverslips, and images were obtained using a confocal microscope (FluoView FV1000; Olympus, Tokyo, Japan).
Oct compound medium
Manufactured by Sakura Finetek
Sourced in United States
The OCT compound medium is a pre-made solution designed for use in cryostat sectioning. It is a water-soluble, inert medium that helps to embed and support tissue samples during the sectioning process. The core function of this product is to facilitate the cutting of thin, consistent tissue slices for microscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fitc and tritc, by Santa Cruz Biotechnology (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Reddot2, by Biotium (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Roti mount fluorcare mounting medium, by Carl Roth (1 mentions)
Dm750 microscope, by Leica (1 mentions)
Vibratome, by Leica (1 mentions)
0.2 m filter, by Cytiva (1 mentions)
Z6100, by Hitachi (1 mentions)
Sigma fast 3 3 diaminobenzidine, by Merck Group (1 mentions)
Anti cd68, by Abcam (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Cm 3050c cryostat, by Leica (1 mentions)
8 protocols using oct compound medium
1
Immunofluorescence Analysis of Murine Brain
2
Immunohistochemical Brain Tissue Processing
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Mice were anesthetized and transcardially perfused with 4% PFA (w/v) in PBS (pH 7.4). Brains were removed, postfixed for 6 h in 4% PFA, cryo‐protected in 30% (w/v) sucrose at 4°C, and embedded in Tissue‐Tek O.C.T. Compound medium (Sakura Finetek). Free‐floating cryosections (40 µm) were washed three times in Tris‐buffered saline (TBS), permeabilized in 0.3% (v/v) Triton X‐100 in TBS for 5 min, and incubated with blocking solution (10% (v/v) normal serum, 1% (w/v) BSA, 0.3% Triton X‐100 in TBS) for 2 h. Sections were incubated with the corresponding primary antibodies at 4°C for overnight. After three washes in 0.025% Triton X‐100 in TBS, sections were incubated with the corresponding secondary antibodies. Sections were counterstained with Hoechst 33258 (H‐3569, Molecular Probes) for 5 min or with RedDot2 (40061, Biotium) for 20 min. After three washes in TBS, sections were mounted with Roti‐Mount FluorCare mounting medium (Roth).
3
Histological Validation of Microneedling
4
Brain Tissue Fixation and Cryosectioning
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For brain tissue collection, the mice were anesthetized and transcardially perfused with saline followed by (4%) paraformaldehyde and then fixed with (4%) paraformaldehyde for 48 h. Further, the brain tissues were immersed in a 20% sucrose solution for 48 h. Next, the Brain were fixed vertically in the OCT compound medium, Sakura Finetek USA, Inc., Torrance, CA, USA). For the brain cross-section (14 µm in size) using a vibratome (Leica, Nussloch, Germany) and stored at −80 °C.
5
Immunohistochemical Brain Tissue Preparation
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For immunohistochemistry (IHC) and morphological analysis, the experimental mice were anaesthetized and transcardially perfused with 0.9% normal saline solution and 4% paraformaldehyde, respectively. The whole brain was removed carefully and fixed at 4 °C in ice-cold paraformaldehyde for 72 h. The brain was placed in 20% sucrose PBS solution for 48 h. Next, the brain was fixed by freezing vertically in optimum cutting temperature (O.C.T) compound (tissue-Tek O.C.T compound medium, Sakura Finetek USA, Inc., Torrance, CA, USA). Then, 14-µm brain coronal sections were taken with a CM3050C cryostat microtome (Leica, Nussloch, Germany) and collected on polarized slides and mounted.
6
Quantification and Localization of Sodium and Potassium Ions in Plants
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Sodium and potassium ions in shoots and roots were quantified by a wet digestion method [90 ]. Dried, finely powered plant samples (50 mg) were digested in HNO3/H2O2 solution (2:1) in a microwave oven for 4–5 min until the solution became clear. The digested solution was shaken gently and filtered through 0.2-µm filters (Whatman, Maidstone, England), and the solid fraction was discarded. The contents of Na+ and K+ in the extract were quantified by atomic absorption spectrophotometry (Z-6100, Hitachi, Tokyo, Japan).
For localization of sodium and potassium ions, we prepared samples according to the protocol of Mitsui et al. [91 (link)]. Harvested basal portions of shoots were immediately frozen and embedded in OCT compound medium (Sakura Finetek USA, Inc., Torrance, CA, USA), which contained 10.24% w/w polyvinyl alcohol, 4.26% w/w polyethylene glycol, and 85.50% w/w of a nonreactive ingredient. Then 5-µm sections were scanned with an electron probe microanalyzer (EPMA-1605; Shimadzu, Kyoto, Japan).
For localization of sodium and potassium ions, we prepared samples according to the protocol of Mitsui et al. [91 (link)]. Harvested basal portions of shoots were immediately frozen and embedded in OCT compound medium (Sakura Finetek USA, Inc., Torrance, CA, USA), which contained 10.24% w/w polyvinyl alcohol, 4.26% w/w polyethylene glycol, and 85.50% w/w of a nonreactive ingredient. Then 5-µm sections were scanned with an electron probe microanalyzer (EPMA-1605; Shimadzu, Kyoto, Japan).
7
Liver Macrophage Immunohistochemistry in Frozen Sections
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The livers were placed into plastic cassettes, mounted with OCT compound medium (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and frozen in liquid nitrogen. Sections were obtained (7 μm) and subjected to haematoxylin and eosin staining for the analysis of liver histology as well as for immunohistochemical study. Liver sections were fixed with methanol/acetone (1:1), permeabilized with 0.02% Tween 20 in phosphate‐buffered saline (PBS‐T) and blocked with 5% goat serum in PBS‐T. Sections were incubated overnight at 4°C with anti‐CD68 (1:200, Abcam, ab 31630, rabbit) to detect liver macrophages. Antibody detection was performed by employing a horseradish peroxidase‐conjugated secondary antibody (goat anti‐rabbit IgG H&L [Biotin] ab97049, 1:500), the VECTASTAIN ABC Kit (Vector Laboratories) and SIGMAFAST 3,3‐diaminobenzidine as substrate (Sigma, St. Louis, MO, USA). Sections were counter‐stained with haematoxylin.
8
Postnatal Day 7 Rat Brain Biochemistry
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On postnatal day 7, Dawley (male) rat pups were sacrificed after 4 h of injection, and the brain was removed immediately for biochemical analysis and stored at −80 °C. For immunoblot analysis, tissues were homogenized in 0.2 M phosphate-buffered saline (PBS) with phosphatase inhibitor and protease inhibitor cocktail. The sample was centrifuged at 13,000 rpm at 4 °C for 20 min, and the supernatant was collected and stored at −80 °C. For immunofluorescence analysis, the animals anaesthetized were perfused transcardially with normal saline solution until the whole blood from the body was removed followed by fixation with 4% paraformaldehyde (PFA). The brain was then removed, fixed in PFA at 4 °C for 72 h and then kept in 20% sucrose solution for 48 h and frozen in O.C.T. (TissueTek O.C.T. Compound Medium, Sakura Finetek USA, Inc., Torrance, CA, USA). For the preparation of brain slices, the coronal plane (14 μm) tissue sections were obtained and thaw-mounted on the gelatin-coated slide using a CM 3050C cryostat (Leica, Germany).
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