Another set of experiments was performed for histological examinations. After euthanasia, the rats were perfused with 10 % formaldehyde neutral buffer solution (Sigma-Aldrich, St. Louis, MO, USA). Then the maxillary bones were excised, and fixed in 10 % formaldehyde neutral buffer solution at 4 °C for 3 days. The bones were decalcified in a rapid decalcification solution, K-CX (Falma, Tokyo, Japan), at 4 °C for 24 h, followed by conventional dehydration and paraffin embedding. After cutting into 5 μm-thick sections, the specimens were deparaffinized and then stained with hematoxylin-eosin (HE) or immunostained with either anti-11β-HSD1 antibody (Bioss, Woburn, MA, USA) or anti-11β-HSD2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) using Envision + kit/HRP (DAB) (Dako, Glostrup, Denmark). Images were obtained using an all-in-one microscope, BZ-9000 (Keyence, Osaka, Japan).
Formaldehyde neutral buffer solution
Manufactured by Merck Group
Sourced in United States
Formaldehyde neutral buffer solution is a laboratory reagent used to preserve and fix biological samples. It provides a neutral pH environment to maintain the structural integrity of cells and tissues during processing and storage.
Automatically generated - may contain errors
Lab products found in correlation
S1700, by Agilent Technologies (2 mentions)
Bz 9000, by Keyence (1 mentions)
Anti 11β hsd2 antibody, by Santa Cruz Biotechnology (1 mentions)
Envision kit hrp dab, by Agilent Technologies (1 mentions)
Silanized slides, by Agilent Technologies (1 mentions)
Nanozoomer digital pathology slide scanner, by Hamamatsu Photonics (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Nichirei Biosciences (1 mentions)
Anti hsv 1 antibody, by Agilent Technologies (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
Lidocaine hcl, by Merck Group (1 mentions)
5 protocols using formaldehyde neutral buffer solution
1
Histological Examination of Maxillary Bones
2
Quantitative Analysis of HSV-1 in Orthotopic Tumors
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Mice harboring orthotopic TE8-luc tumors were treated and euthanized 34 days after the treatment as scheduled in Figure 3 A. Orthotopic tumors were harvested, fixed in 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St Louis, MOSA) for 72 h, and embedded in paraffin. Sections (5-μm thick) were rehydrated using an alcohol gradient and subjected to heat-mediated antigen retrieval using target retrieval solution S1700 (Dako, Santa Clara, CA).
Sections were mounted on silanized slides (Dako Cytomation, Glostrup, Denmark) and stained with HE. Sequential sections were subjected to immunohistochemical analysis to detect HSV-1. The sections were treated with peroxidase blocking solution (Dako) and Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with a rabbit polyclonal anti-HSV-1 antibody (1:2,000 dilution, 3 μg/mL) (Dako Cytomation), rinsed, and incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Nichirei Bioscience, Tokyo, Japan). The sections were developed with 3,3′-diaminobenzidine (DAB) Peroxidase Substrate kit (Vector laboratories, Burlingame, CA), and then counterstained with hematoxylin. A NanoZoomer Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to view slides.
Sections were mounted on silanized slides (Dako Cytomation, Glostrup, Denmark) and stained with HE. Sequential sections were subjected to immunohistochemical analysis to detect HSV-1. The sections were treated with peroxidase blocking solution (Dako) and Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with a rabbit polyclonal anti-HSV-1 antibody (1:2,000 dilution, 3 μg/mL) (Dako Cytomation), rinsed, and incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Nichirei Bioscience, Tokyo, Japan). The sections were developed with 3,3′-diaminobenzidine (DAB) Peroxidase Substrate kit (Vector laboratories, Burlingame, CA), and then counterstained with hematoxylin. A NanoZoomer Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to view slides.
3
Quantifying Tumor-Infiltrating CD8+ T Cells
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Mice were sacrificed on day 14, and tumors were fixed in 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St. Louis, MO, USA) for 72 h and embedded in paraffin. Sections were rehydrated through an alcohol gradient and subjected to heat-mediated antigen retrieval using target retrieval solution S1700 (Dako, Santa Clara, CA, USA). The sections were stained with an anti-mouse CD8 antibody (Abcam, Cambridge, UK) followed by 3,3′-diaminobenzidine as the chromogenic substrate. For semiquantitative analysis, positive cells were counted in five fields/section.
4
Matrigel Invasion Assay with Suramin
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Corning Biocoat Matrigel Invasion Chamber (Corning, Two Oak Park, MA, USA) was used for the invasion assays. HSC‐3 and SAS cells were washed in PBS and then suspended in DMEM without FBS. The cells (1.0 × 105) were added to the upper chamber. The lower chamber was filled with 0, 20, 50, and 100 μmol/L of suramin in DMEM without FBS. After incubation for 24 hours, the cells were fixed with 10% formaldehyde neutral buffer solution (Sigma) for 20 minutes at room temperature, before being stained with crystal violet (0.05% in distilled water) (Katayama Chemical, Osaka, Japan) for 10 minutes. The invading cells were counted using an inverted OLYMPUS CKX41 microscope at ×40 magnification.
5
Bovine blood and liver sampling
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Blood samples were collected between 0700 and 0900 h before feeding for 3 consecutive days from the coccygeal vein and immediately centrifuged at 2,360 × g for 15 min at 4°C. Serum was obtained and stored at -80°C until analysis. After the blood collection on the last day, liver biopsies were taken from the 11th or 12th costal space of the right using an 18-gauge biopsy probe (EMENT-1815; Shenzhen Yiman Technology Co. Ltd.). Before liver biopsy, the intercostal space of cows was shaved before the liver biopsy, sanitized with iodine scrub and 75% alcohol, and anesthetized with a subcutaneous injection of 2% lidocaine HCl (Sigma-Aldrich Co.). A scalpel blade was used to make a 3-mm stab incision in the skin. The liver puncture needle was then inserted through the intercostal muscle and into the liver. Liver tissue biopsies were immediately frozen in liquid nitrogen, and a subsample was fixed with 10% formaldehyde neutral buffer solution (Sigma-Aldrich Co.).
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