Mouse mammary gland was fixed in 10% buffered formalin and processed as hematoxylin and eosin stained slides and unstained formalin fixed paraffin embedded mammary tissue section slides. Collagen was stained using Trichrome Stain Kit according to the manufacturer’s protocol (TRM-1-IFU, ScyTek). Sections of formalin fixed paraffin embedded mouse mammary gland and human breast cancer tissue microarray slides (BRC1501, Pantomics) were deparaffinized and rehydrated. After heat-induced antigen retrieval, sections were incubated with 5% BSA and then incubated with primary antibodies: anti-TET2 (#ABE364, Millipore Sigma, 1:250), anti-Cre (#15036, Cell Signaling, 1:200), anit-Ki67 (#PIPA519462, Invitrogen, 1:200), and anti-ERα (#ab32063, Abcam, 1:100) overnight at 4 °C. Next, appropriate secondary antibody was applied to the section and visualized with DAB chromogen kit (BioCare Medical). Slides were counterstained with hematoxylin and the images were taken by Olympus BX53 Upright Microscope. For human breast cancer tissue microarray, the histological grading and pathological annotation (tumor grade and subtype) were provided by Pantomics. The correlation between the expression levels of the proteins and with the tumor grade was analyzed using Chi-Square test.
Trichrome stain kit
Manufactured by ScyTek Laboratories
Sourced in United States
The Trichrome Stain Kit is a laboratory product designed for the staining of tissue sections. It is used to differentiate various components within a tissue sample, such as collagen, muscle, and nuclei. The kit provides the necessary staining solutions and protocols to facilitate this histological analysis.
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Lab products found in correlation
Bz 9000, by Keyence (2 mentions)
Buprenorphine, by Henry Schein (2 mentions)
Ketamine, by Henry Schein (2 mentions)
Xylazine, by Henry Schein (2 mentions)
Bx53 upright microscope, by Olympus (1 mentions)
Anti erα, by Abcam (1 mentions)
Dab chromogen kit, by Biocare Medical (1 mentions)
Anti cre, by Cell Signaling Technology (1 mentions)
Hematoxylin and eosin stain kit, by Vector Laboratories (1 mentions)
Mayer s hemalum solution, by Merck Group (1 mentions)
Citrate based antigen unmasking solution, by Vector Laboratories (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Envision system hrp labeled polymer, by Agilent Technologies (1 mentions)
Anti rat igg cy3, by Jackson ImmunoResearch (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
12 protocols using trichrome stain kit
1
Immunohistochemical Analysis of Mouse Mammary Gland and Human Breast Cancer
2
Quantifying Lung Fibrosis in Mice
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Mouse lung tissue was fixed with 10% neutral buffered formalin (NBF) for 24h, then embedded in paraffin. The paraffin blocks were sectioned at 5 μm and stained with a hematoxylin and eosin stain kit (Vector laboratories, Newark, CA, USA, H-3502) or Trichrome Stain Kit (ScyTek, TRM-2-IFU) using the manufacturer’s standard procedures. Ashcroft score was used to assess fibrosis (Hubner et al. 2008 (link)). According to the scale defined by Ashcroft et al., the degree of fibrosis region in the lung was classified and analyzed from grade 0 to grade 8.
3
Histological Analysis of Skin Wound Repair
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Skin specimens from the lesion sites were collected at day 21, fixed with 10% formalin, embedded in paraffin, and cut into 6 μm paraffin sections. These were deparaffinized and rehydrated prior to staining. A trichrome stain kit (ScyTek Laboratories, Logan, UT, USA) containing Masson’s trichrome stain was used to observe the collagen fiber deposition. Immunohistochemistry was performed via the ABC method using a Vectastain ABC HRP kit (Vector Laboratories, Burlingame, CA, USA). A citrate-based antigen unmasking solution (Vector Laboratories) was used for antigen retrieval, and an alpha smooth muscle actin (α-SMA) antibody (GTX629702, GeneTex, Hsinchu, Taiwan) was used to target myofibroblasts. Sections were counterstained with Mayer’s hemalum solution (Merck Millipore, Darmstadt, Germany).
4
Histological Analysis of Skin Tissue
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Formalin-fixed sections of uninjured and post-injured skin tissues were deparaffinized by immersion in xylene and then rehydrated. The samples were stained with Trichrome Stain Kit (Modified Masson's) (ScyTek Laboratories, Inc.) For the histological analysis of uninjured skin and measurements of fibrosis in post-injured skin tissues, Image J image analysis software was used (National Institutes of Health (NIH) Image).
5
Quantifying Renal Fibrosis via Trichrome Staining
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The kidneys were fixed by immersion overnight at 4°C in 4% paraformaldehyde and sucrose in phosphate buffered saline (PBS). The kidneys were then placed overnight at 4°C in PBS with 30% sucrose. Thereafter the tissues were frozen in OCT embedding medium (Tissue-Tek, Torrance, CA, USA). To determine the degree of renal fibrosis, five-micrometer frozen sections were prepared and subjected to Masson's trichrome staining using a Trichrome Stain Kit (ScyTek Laboratories, Logan, UT, USA). Ten randomly chosen nonoverlapping fields were collected by a BZ-X710 microscope (Keyence, Osaka, Japan), and fibrotic areas were quantified using the BZ-X Analyzer (Keyence).
6
Bladder and Iliac Artery Histological Analysis
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The bladder and the common iliac artery were harvested from rats at 24 weeks of age and fixed in fresh 4% paraformaldehyde (pH 7.4). Modified Masson's trichrome staining was performed using Trichrome Stain Kit (Scy Tek Laboratories, Logan, UT). IHC staining was performed with same antibodies used in Western blotting. Antigen retrieval was performed in citrate buffer solution (10 mM, pH 7.0) at 70ºC for 60 min. Background activity was blocked with Protein Block (Dako, Glostrup, Denmark) at room temperature for 1 h. Reaction products were visualized by EnVision + System-HRP Labeled Polymer (Dako) and the Liquid DAB + Substrate Chromogen System (Dako). Number of nuclei was counted from Hematoxylin-stained nuclei of lamina propria (urothelium and muscle layer were subtracted) of the whole circumference of the bladder using ImageJ software (https://imagej.nih.gov/ij/ ). IHC staining was quantified by measuring the stain-positive area ratio for all layers of the bladder from each slide using ImageJ software.
7
Immunohistological Analysis of Tissue Samples
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Frozen and paraffin tissues were analyzed histologically as described previously.13 (link) For staining of paraffin tissues with anti-CD3 mAb (Clone: CD3-12, GeneTex), antigen retrieval was conducted by heating sections in 1 mM EDTA solution (pH 9.0) for 15 min in a microwave oven after deparaffinization. The following antibodies and reagents were used for immunohistologic analysis: purified-anti-CD3ε mAb (1:100; 100302, for frozen tissue, BioLegend), purified-anti-I-A/I-E mAb (1:100: 107602, BioLegend), purified-anti-CD3 mAb (1:100; GTX42110, for paraffin tissue, GeneTex), purified-anti-F4/80 mAb (1:100; 123102, BioLegend), Cy3–anti-Armenian hamster IgG (1:200; 127-165-160, Jackson Immunoresearch Laboratories, West Grove, PA, USA), Cy3–anti-rat IgG (1:200; 712-165-153, Jackson Immunoresearch Laboratories), AF488–anti-rat IgG (1:200; A-11006, ThermoFisher Scientific), and BODIPY493/503 (1:1000; D3922, Molecular Probes, Eugene, OR, USA). Masson’s trichrome staining was conducted by using Trichrome Stain Kit (Modified Masson’s, ScyTek Laboratories, Logan, UT, USA), according to the manufacturer’s protocol. Tissue sections were examined under a fluorescence microscope (model BZ-9000, Keyence, Osaka, Japan).
8
Gomori Trichrome Staining Protocol
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Gomori trichrome staining was performed using a Trichrome Stain Kit according to the manufacturer recommendations with modifications (ScyTek Laboratories). Samples were deparaffinized, rehydrated, and incubated for 1 h in Bouin’s solution preheated to 65 °C under a fume hood. Slides were incubated 10 min in Weigert’s iron hematoxylin stain at room temperature. Excess stain was removed by rinsing in water and acid alcohol solution. Samples were next stained with Trichrome stain solution for 20 min at room temperature, followed by washing in water and acetic acid solution. Slides were dehydrated in alcohol and xylol, mounted with RotiHisto Kit mounting medium (Carl Roth, 66381), and observed under Zeiss Axioskop 2 microscope.
9
Quantifying Skin GVHD Severity Post-HSCT
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Recipients were weighed and scored for clinical skin GVHD every week after allo-HSCT. The clinical skin GVHD score was determined as previously described [13] : a healthy appearance was given a score of 0; skin lesions with alopecia ≤1 cm 2 in area, a score of 1; 1-2 cm 2 , a score of 2; 2-5 cm 2 , a score of 3; 5-10 cm 2 , a score of 4; 10-15 cm 2 , a score of 5; 15-20 cm 2 , a score of 6; and >20 cm 2 , a score of 7). Additionally, animals were assigned 0.4 point for skin disease (lesions or scaling) on the tail and 0.3 point each for lesions on ears or paws. Paraffin-embedded skin tissues were stained with hematoxylin and eosin. Masson's staining was performed on Paraffin slides were stained with Masson's trichrome using a Trichrome Stain Kit (ScyTek Laboratories, Logan UT, USA).
10
Masson's Trichrome Staining of Paraffin-Embedded Heart
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The paraffin-embedded heart sections were stained with Masson's trichrome by using a trichrome stain kit (ScyTek Laboratories, Inc, Logan, UT) in accordance with the manufacturer's instructions. The stained sections were observed under fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan), and the infarct and left ventricular areas were assessed using a BZ-II analyzer (Keyence).
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