Tissues were fixed in 4% PFA for 24 h, dehydrated with an ascending ethanol series followed by xylene, and paraffin embedded. Tissues were sectioned at 4–6 µm and collected onto SuperFrost slides (ThermoFisher Scientific). Sections were stained with haematoxylin and eosin (H&E), and with anti-PSA (Ventana Cat #: 760-2506) and anti-Ku70 (ab58150) antibodies. For antibody staining, sections were treated with peroxidase blocking reagent (Dako, S2001) for 5 min at room temperature, antigen-retrieved (10 mM sodium citrate, pH 6.0) at high temperature/pressure for 1 min, and blocked with Protein Block Serum Free Medium (Dako, X0909) for 30 min at room temperature. Primary antibodies (anti-PSA at 1:100, and anti-Ku70 at 1:6000) in 0.5% BSA were incubated on the slides overnight at 4 °C. The DAKO LSAB2 detection system (Cat #K0675) was used per manufacturer’s instruction to secondarily label the primaries. A DAB substrate kit (abcam, K3466) was used to develop the chromogenic signal. Sections were counterstained with Mayer’s hematoxylin, followed by cover-slipping in DePex mounting media (Electron Microscopy Sciences, Fort Washington, PA, USA). Negative controls were prepared by substituting primary antibody with mouse IgG1 (Dako, X0931).
Depex mounting media
Manufactured by Electron Microscopy Sciences
Sourced in United States
DePex is a clear, mounting medium used to affix and preserve specimens for microscopic analysis. It is a solvent-based resin that dries into a transparent, hard film to protect and seal samples mounted on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Dab substrate kit, by Abcam (1 mentions)
Peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Mouse igg1, by Agilent Technologies (1 mentions)
Lsab2 detection system, by Agilent Technologies (1 mentions)
Schiff s reagent, by Merck Group (1 mentions)
Eclipse ti s microscope, by Nikon (1 mentions)
Vectashield hard set mounting medium for fluorescence, by Vector Laboratories (1 mentions)
Vibratome, by Leica (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Merck Group (1 mentions)
Fluoromount, by Electron Microscopy Sciences (1 mentions)
Anti ctip2, by Abcam (1 mentions)
5 protocols using depex mounting media
1
Immunohistochemical Analysis of Prostate Tissues
2
Giemsa Staining of Dry Slides
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Dry slides were stained in Schiff’s reagent (Sigma-Aldrich Inc.) for 1 h, rinsed twice in 2× SSC buffer (trisodium citrate 8.8 g/L, sodium chloride 17.5 g/L), stained in Giemsa (azure-eosin-methylene blue solution for microscopy) (Electron Microscopy Sciences Inc.). 1% Giemsa solution is concentrated Giemsa diluted in Sörenssen’s buffer (disodium hydrogen phosphate dihydrate 11.87 g/L, potassium dihydrogen phosphate 9.07 g/L). Following the rinse in 2XSSC buffer slides were stained in 1% Giemsa for 5 min, then rinsed quickly once in Sörenssen buffer, then in running distilled water and then air-dried. Dry slides were placed in Xylene for 3 h before coverslips were glued with DePex mounting media (Electron Microscopy Sciences).
3
Tissue Preparation for Microscopic Analysis
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Imaging and analysis was performed using a using a Nikon Eclipse Ti‐S microscope. Coverslips were applied with DEPEX Mounting media (Electron Microscopy Sciences), except for Alkaline Phosphatase staining where Vectashield Hard Set Mounting Medium for Fluorescence (Vector) was applied. Soft tissues were stored in 10% neutral buffered formalin, processing was done using a Sakura Tissue‐Tek VIP 6 automated histoprocessor, and paraffin embedding was done using a Sakura Tissue‐Tek TEC 5 paraffin embedding center. A Microm HM315 microtome was used to section tissues at a thickness of 5 µm, which then were floated onto a slide with a water bath at a temperature between 45 and 55°C. Sections were deparaffinized and rehydrated to distilled water through xylene and graded ethanol (100%–70%).
4
Cresyl Violet Staining and Immunocytochemistry of Brain Sections
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Brains were sectioned coronally (50 μm thick) using a vibratome (Leica) and collected
in 12-well plates. Immunocytochemistry was performed on every 12 th tissue
section. Sections were mounted onto glass slides and dried overnight at ambient
temperature. They were then deparaffinized with xylene, hydrated in descending
concentrations of ethanol, rinsed in water, and immersed in 0.5% cresyl violet for
3 hours. After dehydration in ascending ethanol and xylene, the slides were cover
slipped with DEPEX Mounting Media (Electron Microscopy Sciences). Sections were
incubated with 0.01 M sodium citrate, pH 9.0 in an 80° C water bath for 3 hours for
antigen retrieval followed by blocking (PBS, 0.05% BSA, 2% FBS, 1% Triton X-100, and
0.1% saponin). Primary antibodies for profilin1 (1:1000, Sigma), anti-Map2 (1:500;
Millipore), anti-Ctip2 (1:500; Abcam), and anti-Cry-mu (1:200; Atlas Antibodies) were
applied and incubated overnight at 4° C. Secondary antibodies (i.e., 1:500, Alexa-Flour
488 or 647; Invitrogen) were applied in blocking solution for 2 hours at room
temperature in the dark. Sections were mounted and cover slipped with Fluoromount
(Electron Microscopy Sciences). Quantification of pixel (DAB induced brown colour) was
analysed by ImageJ.
in 12-well plates. Immunocytochemistry was performed on every 12 th tissue
section. Sections were mounted onto glass slides and dried overnight at ambient
temperature. They were then deparaffinized with xylene, hydrated in descending
concentrations of ethanol, rinsed in water, and immersed in 0.5% cresyl violet for
3 hours. After dehydration in ascending ethanol and xylene, the slides were cover
slipped with DEPEX Mounting Media (Electron Microscopy Sciences). Sections were
incubated with 0.01 M sodium citrate, pH 9.0 in an 80° C water bath for 3 hours for
antigen retrieval followed by blocking (PBS, 0.05% BSA, 2% FBS, 1% Triton X-100, and
0.1% saponin). Primary antibodies for profilin1 (1:1000, Sigma), anti-Map2 (1:500;
Millipore), anti-Ctip2 (1:500; Abcam), and anti-Cry-mu (1:200; Atlas Antibodies) were
applied and incubated overnight at 4° C. Secondary antibodies (i.e., 1:500, Alexa-Flour
488 or 647; Invitrogen) were applied in blocking solution for 2 hours at room
temperature in the dark. Sections were mounted and cover slipped with Fluoromount
(Electron Microscopy Sciences). Quantification of pixel (DAB induced brown colour) was
analysed by ImageJ.
5
Immunohistochemical Analysis of MAP-2 in Marmoset Brain
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The brain of a second marmoset, case F, was used to gather MAP-2 immunohistochemistry. The brain’s right hemisphere was sectioned at 40 µm on a freezing microtome. A free-floating brain section was submitted to the heat-induced epitope retrieval to enhance immunolabeling by incubating it for 30 min in 10 mM Tris-EDTA buffer (pH 9.5) at 80 °C79 (link). Then the section was incubated for 60 h at 4 °C with a 1:200 dilution of a rabbit anti MAP-2 primary antibody (188 003, Synaptic Systems, Germany) and for 3 h with a 1:250 dilution of a goat anti-rabbit secondary antibody conjugated to DyLight 680 fluorophore (35569, Invitrogen, Waltham, Ma). Finally, the section was mounted on a glass slide, air-dried, and cover-slipped with DEPEX mounting media (13515, Electron Microscopy Sciences, Hatfield, PA).
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