Immunohistochemistry (IHC) of inflammatory cells was performed on three animals per group. Extracted brains were cut into 3-mm thick coronal slices and immersed in a 20% sucrose solution for 2 hrs at 4°C. Brain slices were embedded in OCT (Sakura Finetek Inc., Torrance, CA, USA) and stored at −80°C. For immunostaining, 10 μm coronal sections were obtained by using a cryostat (Tissue-Tek Cryo3; Sakura Finetek Inc.). Sections were further incubated with 3% H2O2 and 10% methanol in PBS, to block endogenous peroxidase. Sections were subsequently incubated overnight at 4°C with primary antibodies against CD68 (ED-1, 1:50 dilution; Abcam) and CD11b (OX42, 1:50 dilution; Abcam), followed by incubation with a biotin-conjugated secondary anti-rabbit antibody (1:200; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hr and streptavidin-conjugated peroxidase (Vecstatin Abc kit, Vector Laboratories Inc.) for 30 min. The slices were developed by incubation (3 min.) with DAB 0.05% (w/v; Dako, Glostrup, Denmark) and visualized under an IX-51 microscope (Olympus Life and Material Science Europe GMBH, Hamburg, Germany), which was attached to a DS-U2 LCD camera (Nikon Instruments Inc., Melville, NY, USA). All slides were evaluated by a blinded, trained observer. Cells were counted in five representative fields of 192 μm2 in the infarcted area.
Tissue tek cryo3
Manufactured by Sakura Finetek
Sourced in Japan
The Tissue-Tek Cryo3 is a cryostat instrument designed for the preparation of frozen tissue sections. It provides precise temperature control and sectioning capabilities to support various histological and pathological applications.
Automatically generated - may contain errors
Lab products found in correlation
Ox 42, by Abcam (1 mentions)
Vecstatin abc kit, by Vector Laboratories (1 mentions)
Biotin conjugated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Agarose type 9 a, by Merck Group (1 mentions)
Anti tf antibodies, by LifeSpan BioSciences (1 mentions)
Tissue tek, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Ov110, by Olympus (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Type 9 a, by Merck Group (1 mentions)
5 protocols using tissue tek cryo3
1
Immunohistochemical Analysis of Inflammatory Cells
2
Cryosectioning and Immunostaining of the Cochlear Apparatus
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The collected temporal bones were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS; pH 7.4) for 2 h, and then harvested the CA under the microscope (SZX9, Olympus, Japan). The fixed specimens were immersed in PBS with 30% sucrose for 6 h, embedded in 5% agarose (type IX-A, Sigma-Aldrich, Ireland) and 20% sucrose in PBS, frozen in n-hexane (−60°C). These specimens were cut vertically into 15 μm thick sections from planum semilunatum to the center of the crista on a cryostat (Tissue-Tek Cryo3, Sakura Finetek, Japan; Kanda et al., 2008 (link)). At intervals of 45 μm, five sections including the center of CA were multiple-immunostained in each experiment and observed under a confocal microscope (A1+, Nikon, Japan; Supplementary Figure S2 ).
3
Immunohistochemical Detection of TF
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After fluorescent imaging using a microscope, human tissues were snap frozen in OCT (Tissue-Tek, Fisher Scientific, Hampton, NH, USA) and stored for future use at −80°C. The tissues were sectioned into 8-micron slides using a cryostat (Tissue-Tek Cryo3, Sakura Finetek, Alphen aan den Rijn, the Netherlands) and stained with a 1:100 dilution of anti-TF antibodies (LifeSpan BioScience) overnight at 4°C using a sandwich approach. After a brief wash with PBS (1×), slide tissues were treated with secondary biotinylated-labeled antibodies (Vectastain) for 30 min. The slides were then treated with ABC reagent (Vectastain Elite ABC kit, Vector Laboratories) for 30 min, after which 3,3-diaminobenzidine (DAB) was added for color development. The slides were examined using white light microscopy (Carl Zeiss Micro-imaging Inc., Jena, Germany).
4
Evaluating ADC Distribution and Localization in Tumor Tissue
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To evaluate the distribution of the ADCs in the tumor tissue, 100 μg (5 mg/kg) of Alexa647‐conjugated anti‐human, anti‐mouse TF and control ADC were injected intravenously into the tail vein. The biodistributions of the ADCs were analyzed by in vivo imaging system OV‐110 (Olympus, Tokyo, Japan) up to 7 days after injection.
To evaluate the localization of the ADCs in the tumor tissue, 300 μg (15 mg/kg) of Alexa647‐conjugated anti‐human, anti‐mouse TF and control ADC were injected intravenously into the tail vein. Twenty‐four hours after the injection, the tumor was excised from the mice under deep anesthesia. The tumor tissues were embedded in an OCT compound (Sakura Finetek, Tokyo, Japan) and quickly frozen in liquid nitrogen. For immunohistochemical analysis, 10‐μm‐thick frozen sections were prepared using a Tissue Tek Cryo3 (Sakura Finetek) and fixed with 4% paraformaldehyde in PBS (Wako) for 30 min at 4°C. After blocking, the sections were incubated with an anti‐CD31 goat antibody for endothelial cells (2 μg/mL; R&D Systems, Minneapolis, MN) for 1 hr at room temperature. After washing with PBS, the sections were incubated with an Alexa488‐conjugated donkey anti‐goat IgG secondary antibody (1 μg/mL; Invitrogen) for 30 min at room temperature and nuclear stained with a DAPI solution.
To evaluate the localization of the ADCs in the tumor tissue, 300 μg (15 mg/kg) of Alexa647‐conjugated anti‐human, anti‐mouse TF and control ADC were injected intravenously into the tail vein. Twenty‐four hours after the injection, the tumor was excised from the mice under deep anesthesia. The tumor tissues were embedded in an OCT compound (Sakura Finetek, Tokyo, Japan) and quickly frozen in liquid nitrogen. For immunohistochemical analysis, 10‐μm‐thick frozen sections were prepared using a Tissue Tek Cryo3 (Sakura Finetek) and fixed with 4% paraformaldehyde in PBS (Wako) for 30 min at 4°C. After blocking, the sections were incubated with an anti‐CD31 goat antibody for endothelial cells (2 μg/mL; R&D Systems, Minneapolis, MN) for 1 hr at room temperature. After washing with PBS, the sections were incubated with an Alexa488‐conjugated donkey anti‐goat IgG secondary antibody (1 μg/mL; Invitrogen) for 30 min at room temperature and nuclear stained with a DAPI solution.
5
Comprehensive Temporal Bone Analysis of GM-treated Animals
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The animals were euthanized with intramuscular ketamine 40 mg/kg and xylazine 10 mg/kg before and at 14 and 28 days after GM treatment. The collected left temporal bones were fixed in 4% paraformaldehyde/phosphate–buffered saline (PBS) (pH 7.4) for 2 h, and then the CAs were harvested under a microscope (SZX9, Olympus, Japan). The fixed specimens were immersed in PBS with 30% sucrose for 6 h, embedded in 5% agarose (type IX–A, Sigma–Aldrich, St. Louis, MO, USA) and 20% sucrose in PBS, then frozen in n–hexane (−60 °C). These specimens were cut vertically into 15 µm thick sections from the planum semilunatum to the center of the crista on a cryostat (Tissue–Tek Cryo3, Sakura Finetek, Tokyo, Japan) [41 (link)]. At intervals of 45 µm, five sections, including the center of CA, were immunostained.
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