TUNEL staining was performed using an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), according to previously described methods [15 (link),17 (link)]. The sections were postfixed in ethanol-acetic acid (2:1), and then were rinsed. The sections were then incubated with proteinase K (100 μg/mL), rinsed, incubated in 3% H2O2, and permeabilized with 0.5% Triton X-100. After the sections were rinsed again, the sections were incubated in the TUNEL reaction mixture. After the sections were rinsed, the sections were visualized using Converter-POD with 0.03% DAB. Mayer hematoxylin (DAKO, Glostrup, Denmark) was used as a counterstain. The sections were mounted onto gelatin-coated slides, and the coverslips were mounted using Permount (Fisher Scientific).
Mayer hematoxylin
Manufactured by Agilent Technologies
Sourced in Denmark
Mayer hematoxylin is a laboratory stain used in histology and cytology to visualize nuclei and cellular structures. It is a hematoxylin-based stain that produces a blue-purple color when applied to tissue or cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Permount, by Thermo Fisher Scientific (3 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Streptavidin horseradish peroxidase, by Biocare Medical (2 mentions)
Goat anti rabbit biotinylated igg, by Biocare Medical (2 mentions)
Anti aqp2 antibody, by Santa Cruz Biotechnology (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Agilent Technologies (1 mentions)
Hematoxylin eosin he, by Merck Group (1 mentions)
Anti rabbit secondary antibody, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Bx43 microscope, by Olympus (1 mentions)
Ba 2000, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
6 protocols using mayer hematoxylin
1
TUNEL Staining for Apoptosis Detection
2
Histochemical Analysis of Kidney Cysts
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Isolated kidneys were fixed in 40% Formalin and routinely embedded, cut at 4 μm slices, dried and deparaffinized for subsequent histochemistry. Hematoxylin and Eosin (H&E) staining was used to assess kidney morphology. The kidney slices were scanned with Nikon (Melville, NY) Super CoolScan 9000 interfaced with NikonScan 4 software. Further analysis of cyst area was performed using color threshholding method using Metamorph (Molecular Devices, Sunnyvale, CA) software as previously described (33 (link)). For immunohistochemistry, tissue sections were incubated with anti-β-ENaC antibodies (StressMarq, Victoria, Canada) or anti-AQP2 antibodies (Santa Cruz Biotechnology, Dallas, TX). Secondary detection was performed with goat anti-rabbit biotinylated IgG (Biocare, Concord, CA) followed by streptavidin horseradish peroxidase (Biocare) and visualized with DAB (DAKO). All slides were counterstained with a Mayer hematoxylin (DAKO, Carpinteria, CA), dehydrated, and mounted with permanent mounting media (Sakura, Torrance, CA).
Captured images were analyzed with ImageJ software (NIH, Bethesda, MD) to measure signal intensities (arbitrary units, ranged from 0 to 255) in tubules and cysts. Intensities from 7–18 individual tubules or cysts were collected each animal; total number of animals per group was three.
Captured images were analyzed with ImageJ software (NIH, Bethesda, MD) to measure signal intensities (arbitrary units, ranged from 0 to 255) in tubules and cysts. Intensities from 7–18 individual tubules or cysts were collected each animal; total number of animals per group was three.
3
Histological Evaluation of Bone Regeneration
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The parietal calvaria that included the whole defect site (region of interest) was excised and fixed in 10% phosphate-buffered formalin for histological evaluation. The samples were then decalcified using 10% EDTA for two weeks followed by embedding in paraffin blocks. Transverse sections of eight -μm-thickness were made from paraffin blocks using a tissue microtome and stained with hematoxylin-eosin (HE; Sigma, St. Louis, MO).
Immunohistochemical analysis for osteocalcin and osteopontin was performed using anti-rat antibodies. Briefly, the tissue specimens were incubated in a 1:100-diluted anti-osteocalcin antibody solution (Cat no: PA596529) or 1:100 diluted anti-osteopontin antibody solution (Cat no: PA534579) at 37°C for 1 h, followed by 3 washes in phosphate-buffered saline (Sigma, USA). The specimens were further incubated with an anti-rabbit secondary antibody (Abcam, USA). Then, 3,3′-diaminobenzidine tetrahydrochloride (DAB; Dako) was used as a substrate, and the sections were counterstained with Mayer hematoxylin (Dako) and observed under an Olympus BX43 microscope.
Immunohistochemical analysis for osteocalcin and osteopontin was performed using anti-rat antibodies. Briefly, the tissue specimens were incubated in a 1:100-diluted anti-osteocalcin antibody solution (Cat no: PA596529) or 1:100 diluted anti-osteopontin antibody solution (Cat no: PA534579) at 37°C for 1 h, followed by 3 washes in phosphate-buffered saline (Sigma, USA). The specimens were further incubated with an anti-rabbit secondary antibody (Abcam, USA). Then, 3,3′-diaminobenzidine tetrahydrochloride (DAB; Dako) was used as a substrate, and the sections were counterstained with Mayer hematoxylin (Dako) and observed under an Olympus BX43 microscope.
4
Visualization of Cell Death in Colonic Tissue
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TUNEL staining was done by the In Situ Cell Death Detection Kit® (A23210, Roche, Mannheim, Germany) for the visualization of cell death in colonic tissue, according to the method described earlier [50 (link)]. Paraffin slides containing colonic sections were deparaffinized and rehydrated by xylene and ethanol. These sections were washed and then post-fixed using ethanol and acetic acid (2:1) solution. These sections were then treated by proteinase K (100 μg/mL), treated in 3% H2O2, permeabilized with 0.5% Triton X-100, and treated with TUNEL reaction mixture. After washing, these sections were visualized by Converter-POD with 0.03% 3,3′-diaminobenzidine. After counterstaining with Mayer hematoxylin (DAKO), these sections were mounted on gelatin-coated slides, air-dried overnight at room temperature, and next coverslipped by Permount® (Thermo Fisher Scientific).
5
Immunohistochemical Analysis of Apoptotic Markers
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Immunohistochemistry for cleaved caspase-3 and caspase-9 was done according to the method described earlier [51 (link)]. Paraffin slides containing colonic sections were deparaffinized by xylene and ethanol and rehydrated. These sections were washed, and after boiling 10 mM citric acid (pH 6.0) for 10 min, these sections denatured. These sections were treated with mouse anti-cleaved caspase-3 (9661S) and anti-cleaved caspase-9 antibodies (9507S, Cell Signaling Technology Inc., Danvers, MA, USA) diluted 1:200 overnight. These sections were treated with biotinylated anti-rabbit (BA-1000) and anti-mouse secondary antibodies (BA-2000, Vector Laboratories, Burlingame, CA, USA) for 1 h. These sections were then treated with avidin-biotin-peroxidase complex (PK4010, Vector Laboratories) at room temperature for 1 h. By treating these sections with the solution containing 0.05% 3,3-diaminobenzidine and 0.01% H2O2 in 50 mM Tris-buffer (pH 7.6) for approximately 3 min, immunoreactivity was visualized. After counterstaining with Mayer hematoxylin (DAKO), these sections were mounted on gelatin-coated slides, air-dried overnight at room temperature, and coverslipped by Permount® (Thermo Fisher Scientific).
6
Histochemical Analysis of Kidney Cysts
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