IHC was also used for localization of biomarkers and to detect differentially expressed proteins when autofluorescence and light scattering interfered with image quality. IHC was used for DLX5, DLX6, HAND2 and P53. For IHC, sections were deparaffinized and rehydrated in a series of ethanol dilutions. The sections were boiled for 30 seconds in 10% blocking solution (Biocare, MX, DV-200), washed with 0.05% PBS-Triton, then incubated with the following primary antibodies: rabbit polyclonal anti-DLX5 (Abcam, MA, YH05211C), polyclonal goat anti-DLX6 (Santa Cruz, CA, sc-18154), monoclonal mouse anti-HAND2 (Santa Cruz, CA, sc-130629), monoclonal mouse anti-P53 (Abcam, MA, GR1277-1), diluted in horse serum overnight at 4 °C. Next day, they were washed with 0.05% PBS-Triton and then incubated with the secondary anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG (Vector lab PK-7800, CA) for 20 minutes at room temperature and then washed with 0.05% PBS-Triton. We then treated the samples with tertiary antibody streptavidin-peroxidase complex (Vector lab SK-4800, CA) for 5 minutes at room temperature. Finally, the sections were treated with chromagen (Impact Novared Kit SK-4805) followed with hematoxylin (Fisher scientific, 123022) for nuclear staining. The hematoxylin was rinsed off with distilled water and the slides mounted with cytoseal (Fisher Scientific, PA).
Hematoxylin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, China, Germany
Hematoxylin is a staining dye commonly used in histology and cytology. It is a natural dye extracted from the heartwood of the Logwood tree (Haematoxylum campechianum). Hematoxylin is primarily used to stain cell nuclei, allowing for the visualization of cellular structures and tissue architecture under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Eosin y, by Thermo Fisher Scientific (13 mentions)
Permount, by Thermo Fisher Scientific (8 mentions)
Harris modified hematoxylin, by Thermo Fisher Scientific (7 mentions)
Dab chromagen, by Agilent Technologies (7 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (6 mentions)
Vectastain elite abc kit, by Vector Laboratories (6 mentions)
Xylene, by Thermo Fisher Scientific (6 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (6 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
Ds fi1, by Nikon (5 mentions)
Oil red o, by Merck Group (5 mentions)
Abc substrate kit, by Vector Laboratories (5 mentions)
Abc reagent, by Vector Laboratories (4 mentions)
Permount mounting media, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Dab reaction, by Vector Laboratories (4 mentions)
Vectastain elite abc hrp kit, by Vector Laboratories (4 mentions)
Eosin y, by Merck Group (4 mentions)
Diva decloaker, by Biocare Medical (3 mentions)
Cas block, by Thermo Fisher Scientific (3 mentions)
269 protocols using hematoxylin
1
Immunohistochemistry for Protein Localization
2
Immunohistochemical Evaluation of Cell Proliferation
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Cell proliferation (Ki-67) was evaluated by immunohistochemistry in F1 mammary gland and mammary tumors transplants as well as F2 mammary tumors. Brie y, tissues were xed in 10% buffered formalin, embedded in para n, and sectioned (5 µm). Sections were depara nized with xylene and rehydrated through a graded alcohol series. Antigen retrieval was performed by immersing the tissue sections at 98 °C for 40 minutes in 1X Diva Decloaker (Biocare). Tissue sections were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 minutes and were incubated with the primary antibody, overnight at 4 °C. After several washes, sections were treated to the appropriate HRP labeled polymer for 30 min and DAB chromagen (Dako) for 5 minutes. Slides were counterstained with Hematoxylin (Fisher, Harris Modi ed Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. The sections were photographed using an Olympus IX-71 Inverted Epi uorescence microscope at 40x magni cation. Proliferation index (Ki-67 staining) was determined by immunoRatio, a plugin Image J software (National Institute of Health, Bethesda, MD, USA), to quantify Hematoxylin and DAB-stained cells. Differences between groups were analyzed using one-way ANOVA, followed by posthoc analyses.
3
GFAP Immunohistochemistry Protocol
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Sections were prepared in the same way as for the human immunofluorescence staining and were then incubated overnight in the primary antibody for GFAP (1:1000, abcam, ab7260). The following day, sections were washed three times in PBST before being incubated with a biotinylated secondary antibody for 2 h. Sections were then incubated in ABC solution (Vector Laboratories PK-6100) for 1 h at room temperature, before being rinsed 3 times with PBST. Di-aminobenzidine (DAB) solution (Vector Laboratories SK-4100) was used for immunohistochemical development and was applied to the tissue for approximately 30 s. The sections were then rinsed with dH20, and counterstained with filtered Hematoxylin (Thermo Scientific 6765004) for 30 s. Excess Hematoxylin was removed by dipping the sections in 1% acid alcohol. Finally, sections were dehydrated in dH20, 70% EtOH, 90% EtOH and 100% EtOH for 10 min each. Sections were then left in 100% xylene overnight, before being mounted with DPX mounting medium (Thermo Scientific LAMB/ DPX).
4
Immunohistochemistry of Cultured Epithelial Cells
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The mouse anti-human ABCG2 (used at 1:20), mouse anti-human cytokeratin 3+12 antibody (used at 1:50), and rabbit anti-human connexin 43 (used at 1:100) were obtained from Abcam (Cambridge, MA). The ready-to-use rabbit anti-human cytokeratin 4 and mouse anti-human cytokeratin 13 antibodies were obtained from BioGenex (Fremont, CA). Cultured epithelial cells on the amniotic membrane were subjected to immunohistochemistry. Briefly, the amniotic membrane embedded cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Steinheim, Germany) and blocked for endogenous peroxidase using 3% H2O2 in methanol for 20 min. Nonspecific sites were blocked using peroxidase-blocking solution buffer for 15 min (Dako REAL, Glostrup, Denmark) after which the cells were incubated overnight at 4 °C with the primary antibody. Detection of the bound antibody was performed using EnVision Detection Systems Peroxidase/DAB Rabbit/Mouse (Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Cells were counterstained with hematoxylin (Fisher Scientific, Waltham, MA) mounted in a Distrene, Plasticiser, Xylene (DPX; Merck, Darmstadt, Germany) mounting medium, and observed under a light microscope (Nikon, Eclipse E200, Tokyo, Japan) using software (NIS Element D, Tokyo, Japan).
5
Immunohistochemical Staining of Brain Sections
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On PSDs 1–3, brains were removed, flash frozen and stored at −80℃ until use. Brains were cut into 20 -µm sections using a cryostat and mounted onto slides. Sections were fixed with ice cold acetone prior to incubating in blocking buffer (5% BSA in phosphate buffered saline (PBS) with 0.1% Triton X-100) for 1 h at room temperature. The sections were then incubated overnight at 4℃ in the primary antibody against IgG (1:1000; Life Technologies). Sections were washed and incubated with a secondary antibody (HRP anti-mouse, Life Technologies) for 1 h at room temperature. Sections were washed and incubated with DAB (Vector Labs, Burlingame, CA, USA) for 1 h prior to counterstaining with hematoxylin (Fisher Scientific, USA). Alternatively, sections were stained with primary antibodies against fluorescein isothiocyanate (FITC)-conjugated-tomato lectin (1:200; Vector Labs), glial fibrillary acidic protein (GFAP) (1:500, Sigma), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Apoptag Fluorescien kit, Millipore), or cresyl violet (Sigma) overnight at 4℃. Slides were then coverslipped with fluorescent mounting media (Vector Labs) or xylene-based mounting media (Sigma) and images were captured using a Nikon Eclipse Ti microscope and software (Nikon, Melville, NY, USA).
6
Histological Analysis of Tibialis Anterior Muscle
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TA muscle sections (5 µm) were deparaffinized in xylene and fixed in 100% ethanol. Following a rinse in water, samples were stained in hematoxylin (Fisher) for 3 min, rinsed in water, dipped 40 times in a solution of 0.02% HCl in 70% ethanol and rinsed in water again. The sections were stained in a 1% eosin solution (BDH) for 1 min, dehydrated in ethanol, cleared in xylene and mounted with Permount (Fisher). Images were taken with a Zeiss Axioplan2 microscope, with a 20× objective. Quantitative assays were performed on three mice for each genotype and five sections per mouse. The area of muscle fiber within designated regions of the TA muscle sections was measured using the Image J software.
7
Frozen Tissue Immunostaining and Paraffin Sectioning
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For frozen embedding, tissues were fixed in 4% paraformaldehyde (PFA, Sigma) for overnight at 4°C with gentle rotation, immersed in 30% sucrose for cryoprotection then embedded in OCT. Tissue sections were permeabilized with 0.1% Triton X-100 in PBS (PBT), blocked with Cas block (Life Technologies) and immunostained with antibodies against phospho-S6 (1:250; Cell Signaling; cat. no 5364), MAP2 (1:500; Millipore; cat. no. MAB3418), reelin (1:200; Abcam; cat. no. ab78540), CTIP2 (1:500; Abcam; cat. no. ab81465), SATB2 (1:500; Abcam; ab51502) and FOXG1 (1:200, Abcam; cat. no. ab18259). For BrdU (1:50; BD Biosciences; cat. no. 347580) antibody staining, tissues were treated with HCl (2 N) before blocking. Paraffin embedded tissues were sectioned, rehydrated and retrieved for antigen (citrated buffer, pH 6.0) before immunostaining for GFP (1:200; Rockland; cat. no. 600-106-215) followed by detection by DAB (Vector Labs). Hematoxylin (Fisher) counter staining was performed according to the manufacturer’s protocol.
8
Photoreceptor Quantification in Retinal Sections
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The orientation marked eyes were fixed in 4% paraformaldehyde overnight followed by paraffin embedding. Six-micrometer paraffin sections were obtained using a microtome (Shandon AS325, Thermo Scientific, Cheshire, England). After deparaffinization, sections were stained with hematoxylin (Fisher Scientific, Hercules, CA) and eosin (Fisher Scientific, Hercules, CA). Only sections crossing the optic nerve were used and images were captured with Leica DM6000 microscope. Four nonoverlapping sections crossing the optic nerve from each eye were used for photoreceptor counts. The total number of photoreceptors in the whole retinal section was counted in a masked fashion.
9
Antibody Staining Optimization Protocol
10
Immunohistochemical Staining of CD4+ T Cells
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