Toluidine blue stock solution composed of 1g of Toluidine blue O (Sigma-Aldrich) mixed in 100 ml of 70% alcohol. Finally, sections were dehydrated with increasing concentrations of absolute alcohol, cleared in xylene, and mounted in Richard-Allan Scientific Cytoseal™ 60 mounting medium. Labeled mast cells were visualized by use of a Nikon eclipse E600 microscope and images were captured with a Nikon DXM 1200 digital camera under the same settings. Five HPF images were analyzed from each tissue section and the average number of mast cells was assayed.
Cytoseal 60 mounting medium
Manufactured by Thermo Fisher Scientific
Cytoseal 60 is a mounting medium used for microscopy applications. It is a resin-based formulation designed to provide a clear, durable seal for coverslips on prepared slides.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e600 microscope, by Nikon (3 mentions)
Sk 4100, by Vector Laboratories (3 mentions)
Ab16669, by Abcam (3 mentions)
Streptavidin horseradish peroxidase, by Vector Laboratories (3 mentions)
Ab183685, by Abcam (3 mentions)
Sa 5704, by Vector Laboratories (3 mentions)
Biotin conjugated secondary antibody, by Vector Laboratories (3 mentions)
Eclipse 80i microscope, by Nikon (3 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (3 mentions)
Vectastain abc kit, by Vector Laboratories (3 mentions)
Mhs16, by Merck Group (2 mentions)
Sp 5030, by Vector Laboratories (2 mentions)
M 15704, by Thermo Fisher Scientific (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Ab15580, by Abcam (2 mentions)
Dab kit, by Vector Laboratories (2 mentions)
Ba 1000, by Vector Laboratories (2 mentions)
Toluidine blue, by Merck Group (1 mentions)
Dxm1200, by Nikon (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
21 protocols using cytoseal 60 mounting medium
1
Toluidine Blue Staining of Mast Cells
2
Quantifying Muscle Glycogen Content
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Periodic acid Schiff’s (PAS) staining for glycogen was done on fresh frozen 10-μm-thick sections from quadriceps muscle as previously described [30 (link)]. In brief, sections were fixed in Carnoy’s fixative (60% ethanol, 30% chloroform, 10% acetic acid) for 5 min, followed by rinsing three times with water. Sections were immersed in 0.5% periodic acid (Sigma P7875) for 5 min, followed by rinsing with water four times and then transferred to Schiff’s solution (Sigma 3952016) for 10 min, followed by rinsing in running tap water for 10 min. Sections were mounted using Cytoseal 60 mounting medium (Richard-Allan Scientific 8310-16).
Total glycogen content in muscle was quantified using an adaptation of a previously described method [31 ]. In brief, 30 mg of frozen gastrocnemius muscle was dissolved in 1 ml 5 N KOH in a boiling water bath, then 0.2 ml of saturated sodium sulfate and 1.5 ml ethanol were added. Samples were spun at 2,000 × g for 10 min at 4°C and the pelleted material was resuspended in 0.5 ml 2 N HCl and incubated in a boiling water bath for 2 to 2.5 h. Samples were cooled and neutralized to pH 6 to 8 with 4 N KOH in 0.1 M triethanolamine. Glycogen content was determined by measuring the glucose released from glycogen using a glucose assay kit as recommended by the manufacturer (Sigma GAHK20).
Total glycogen content in muscle was quantified using an adaptation of a previously described method [31 ]. In brief, 30 mg of frozen gastrocnemius muscle was dissolved in 1 ml 5 N KOH in a boiling water bath, then 0.2 ml of saturated sodium sulfate and 1.5 ml ethanol were added. Samples were spun at 2,000 × g for 10 min at 4°C and the pelleted material was resuspended in 0.5 ml 2 N HCl and incubated in a boiling water bath for 2 to 2.5 h. Samples were cooled and neutralized to pH 6 to 8 with 4 N KOH in 0.1 M triethanolamine. Glycogen content was determined by measuring the glucose released from glycogen using a glucose assay kit as recommended by the manufacturer (Sigma GAHK20).
3
Hematoxylin and Eosin Staining Protocol
4
Immunohistochemical Staining of Airway Nerves and Eosinophils
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Isolated lungs were fixed in zinc‐buffered formalin (Anatech Ltd., Battle Creek, MI) overnight at 4°C, then stored in 70% ethanol. Transverse sections from left upper and lower lobes were paraffin‐embedded and cut into 10 μm sections. Slides were dewaxed in xylene overnight and rehydrated in 100%, 70%, and 50% ethanol. Antigen retrieval was performed using Antigen Unmasking Solution (Vector, H‐3300, Burlingame, CA). Endogenous peroxidase activity was quenched with 3% H2O2 in cold methanol (−20°C) and slides were permeabilized in Trypsin (Invitrogen, Carlsbad, CA) for 10 mins at 37°C. DNA was denatured using 2N HCl (30 min) and slides were blocked in 10% normal goat serum (Vector S‐1000, Burlingame, CA). Airway nerves were labeled with pan‐neuronal marker mouse anti‐PGP9 primary antibody (1:250 dilution 4°C overnight; ABD Serotech, Oxford, UK). Goat anti‐mouse biotinylated secondary antibody (Invitrogen) was then applied (1:400 dilution at room temp for 2 hours; Vector, VA‐9200, Burlingame, CA) followed by incubation in Vectastain Elite ABC (Vector, PK‐6100, Burlingame, CA) for 30 mins at room temperature. Slides were developed with Vector SG (Vector). Eosinophils were stained with 1% Chromotrope 2R (Sigma, St. Louis, MO) for 1 min at room temperature. Slides were allowed to dry and mounted in Cytoseal 60 mounting medium (Richard‐Allan Scientific, San Diego, CA).
5
Semi-quantifying Anti-NMDAR1 Autoantibodies
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Immunohistochemistry (IHC) was conducted as previously described (Ji et al., 2013 (link); Kim et al., 2012 (link)) to semi-quantify the levels of blood anti-NMDAR1 autoantibodies in individual mice. Wildtype mouse brain paraffin sections were used for IHC analysis with a dilution of mouse serum at 1:200 with antibody diluent solution (DAKO, S080983–2). Mouse anti-NMDAR1 monoclonal antibody (BD, cat. 556308) was used as the positive control with a dilution at 1:40,000. ImmPRESS peroxidase-micropolymer conjugated horse anti-mouse IgG (H + L) (Vector Labs, MP-7402) was used as the secondary antibody. Biotinylated goat anti-human IgG (H + L) secondary antibody was used for detection of human plasma anti-NMDAR1 autoantibodies. Chromogenic reaction was conducted with ImmPACT NovaRED Peroxidase Substrate (Vector Labs, SK-4805). Slides were mounted with Cytoseal 60 mounting medium (Richard-Allan Scientific, 8310–16). Image J was used to measure differential optical intensities between hippocampal CA1 st oriens and corpus callosum to semi-quantify the levels of anti-NMDAR1 autoantibodies.
6
Quantifying Collagen Deposition via Histochemistry
7
Picrosirius Red Staining of Muscle
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Muscle sections were stained with Picrosirius red solution (0.1% in picric acid, 15 min), washed with acetic acid (1%, 3 times), and mounted with Cytoseal 60 Mounting Medium (Thermo Fisher Scientific).
8
Immunohistochemical Analysis of p-ERK1/2 in Pancreas
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Pancreas were fixed in 10% buffered formalin overnight at 4°C, processed and embedded using routine methods. Paraffin sections were deparaffinized, rehydrated, and heated for 12 minutes at 95°C in 10 mmol/L (pH 6) citrate buffer (M-15704, Thermo Fisher Scientific). Afterward, sections were incubated with 3% hydrogen peroxide (59105926, Millipore corporation) for 10 minutes and blocked-in animal-free Blocker (SP-5030, Vector laboratories) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibody against p-ERK1/2 (1:200 dilution, Cell Signaling Technology, catalog no. 4376, RRID:AB_331772). The following day, paraffin sections were incubated with biotin-conjugated secondary antibody for 1 hour at room temperature (856743, Life Technologies), horseradish peroxidase (HRP) streptavidin for 1 hour at room temperature (856743, Life Technologies), and developed by DAB (SK-4100, Vector Laboratories), followed by hematoxylin (MHS16, Sigma) staining. Sections were then dehydrated, mounted in Cytoseal 60 mounting medium (8310-16, Thermo Fisher Scientific), and analyzed using an Olympus BX51 microscope. Scoring: Five or more fields per sample (at magnification × 200) were scored and the percent of positive cells was calculated as described previously (23 (link)).
9
Immunohistochemical Analysis of SSTR2 Expression
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Tissues
of interest (e.g., tumors, suspicious lesions, NET-associated organs)
were embedded in paraffin or OCT to prepare formalin-fixed, paraffin-embedded
(FFPE), or frozen sections, respectively. Blocks were then serially
sectioned at 5 μm thickness, and one section per block was stained
with standard hematoxylin and eosin (H&E). Immunohistochemistry
(IHC) staining was performed on adjacent sections as we previously
described.17 (link),21 (link) Briefly, after peroxidase inactivation,
sections were incubated with anti-SSTR2 rabbit monoclonal antibody
(ab134152, Abcam) overnight at 4 °C. After PBS washing, a secondary
antibody (biotinylated goat anti-rabbit-polyvalent IgG) was applied
for 10 min at room temperature. For visualization, a DAB detection
kit (ab64261, Abcam) was used according to the manufacturer’s
instructions. Sections were then counterstained with Mayer’s
hematoxylin (Fisher Healthcare), dehydrated through two changes of
alcohol, cleared in xylene, and cover-slipped with Cytoseal 60 mounting
medium (Thermo Scientific). For FFPE sections, the slides were deparaffinized
before H&E and IHC staining, and antigen retrieval was performed
prior to IHC staining. For mesoscopic imaging, an adjacent section
from each tissue was scanned on an Odyssey (LI-COR) at 800 nm with
the highest resolution (21 μm).
of interest (e.g., tumors, suspicious lesions, NET-associated organs)
were embedded in paraffin or OCT to prepare formalin-fixed, paraffin-embedded
(FFPE), or frozen sections, respectively. Blocks were then serially
sectioned at 5 μm thickness, and one section per block was stained
with standard hematoxylin and eosin (H&E). Immunohistochemistry
(IHC) staining was performed on adjacent sections as we previously
described.17 (link),21 (link) Briefly, after peroxidase inactivation,
sections were incubated with anti-SSTR2 rabbit monoclonal antibody
(ab134152, Abcam) overnight at 4 °C. After PBS washing, a secondary
antibody (biotinylated goat anti-rabbit-polyvalent IgG) was applied
for 10 min at room temperature. For visualization, a DAB detection
kit (ab64261, Abcam) was used according to the manufacturer’s
instructions. Sections were then counterstained with Mayer’s
hematoxylin (Fisher Healthcare), dehydrated through two changes of
alcohol, cleared in xylene, and cover-slipped with Cytoseal 60 mounting
medium (Thermo Scientific). For FFPE sections, the slides were deparaffinized
before H&E and IHC staining, and antigen retrieval was performed
prior to IHC staining. For mesoscopic imaging, an adjacent section
from each tissue was scanned on an Odyssey (LI-COR) at 800 nm with
the highest resolution (21 μm).
10
Quantifying Mitral Cell Density in Olfactory Bulb
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To measure the density of mitral cells in the OB, we stained sections with Cresyl violet (one section every eighth section). Slides were incubated for 9 min in 0.1% Cresyl violet solution, and were dipped into 95%, 100% ethanol and xylene before coverslipping with Cytoseal 60 mounting medium (Thermo Fisher Scientific). On blind coded sections stained with Cresyl violet, we measured the density of mitral cells in the mitral cell layer of the OB. Mitral cells can be easily identified from other cells based on their morphology (large nucleus and cytoplasm). Quantifications were done on a computer-assisted mapping and cell quantification program (Stereo Investigator; MBF Bioscience) coupled to an Imager M2 microscope (ZEISS). We analyzed four sections per animal spaced by 450 µm, and distributed along the rostrocaudal axis at equivalent locations in each animal, in four to five animals per group. We outlined the mitral cell layer and counted every mitral cell in that area. We then calculated the density of cells per section (number of cells per surface area) and the mean density per animal. We then calculated the mean density per group and analyzed the data by Kruskal–Wallis testing with post-hoc Dunn’s multiple comparison analysis using Prism 6.0 (GraphPad Software).
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