TG were fixed in 10% neutral buffered formalin, paraffin embedded, and then sectioned at 4 μm thickness. For mCherry staining, antigen retrieval was performed for 12 hours at pH 9 in a 65°C water bath using Tris-EDTA buffer. Sections were then stained with a mouse monoclonal anti-mCherry antibody (1:1,000) provided by FHCRC Experimental Histopathology (Benjamin Hoffstrom), and detection was performed using PowerVision poly-HRP anti-mouse (Leica), along with the chromagen DAB (Dako) 2 times for 4 minutes. Slides were then counterstained with hematoxylin (BioCare) and Tacha's bluing solution at 25% for 2 minutes. Isotype controls of mouse Ig were used as negative control. For NeuN staining, antigen retrieval was performed as above for 12 hours at pH9 in a 65°C water bath. Sections were then stained with a mouse monoclonal anti-NeuN antibody (clone A60, Millipore mab377, 1:800), and detection was performed using PowerVision poly-HRP anti-rabbit and the Mouse on Mouse Basic Kit (Vector Laboratories, catalog BMK-2202).
Hematoxylin
Manufactured by Biocare Medical
Sourced in United States
Hematoxylin is a histological stain commonly used in medical laboratories. It is a natural dye extracted from the logwood tree and is used to stain cell nuclei blue, providing contrast for microscopic analysis of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Biotinylated secondary antibody, by Biocare Medical (2 mentions)
Dako autostainer plus, by Agilent Technologies (2 mentions)
Streptavidin horseradish peroxidase, by Thermo Fisher Scientific (2 mentions)
Species appropriate irrelevant igg, by Jackson ImmunoResearch (2 mentions)
Vina green chromogen, by Biocare Medical (2 mentions)
Universal negative, by Biocare Medical (2 mentions)
Gapdh polyclonal antibody, by Cell Signaling Technology (2 mentions)
Background sniper, by Biocare Medical (2 mentions)
Vectastain elite dab kit, by Vector Laboratories (2 mentions)
Mouse on mouse basic kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine substrate, by Thermo Fisher Scientific (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Chrompure mouse igg, by Jackson ImmunoResearch (1 mentions)
Dm4000b microscope, by Leica (1 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Bloxall endogenous blocking solution, by Vector Laboratories (1 mentions)
Rodent ap polymer, by Biocare Medical (1 mentions)
Ecomount, by Biocare Medical (1 mentions)
Proteinase k, by Merck Group (1 mentions)
22 protocols using hematoxylin
1
Immunohistochemical Staining of mCherry and NeuN
2
Immunohistochemical Staining of Tissue Sections
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Four micron thick, formalin fixed, paraffin embedded tissue sections were prepared and immunohistochemistry was carried out on a Dako Plus autostainer (DAKO, Inc, Carpenteria, CA) as previously described [23 (link)]. Briefly, following antigen retrieval and blocking steps, sections were incubated in primary antibody for 60 mins, followed by appropriate biotinylated secondary antibody (Biocare Medical, Conrad, CA), and then streptavidin-horseradish peroxidase (Thermo Scientific Lab Vision). Color was developed with 3,3′-diaminobenzidine substrate (Thermo Scientific Lab Vision) and sections were counterstained with hematoxylin (Biocare Medical, LLC). As a negative control, adjacent serial sections were incubated with species appropriate irrelevant IgG (Jackson ImmunoResearch Labs) at the same concentration as primary antibodies. Similar to the lung tumor burden evaluation described above, mean percentage of positive staining ± SEM was determined by 2 different observers, each calculating the ratio of positive cells/total number of cells × 100 for 4 separate high power fields (63X objective) from at least 2 different mice per group.
3
Immunohistochemical Analysis of CRISPLD2 Expression
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Uterine sections from paraffin-embedded tissue were cut at 6 µm and mounted on silane-coated slides, deparaffinized and rehydrated in a graded alcohol series. Sections were preincubated with 10% normal goat serum in PBS (pH 7.5) and then incubated with primary antibody diluted in 10% normal goat serum in PBS (pH 7.5) overnight at 4°C at 1∶2000 dilution for CRISPLD2 (HPA030055; Sigma Aldrich, St. Louis, MO). On the following day, sections were washed in PBS and incubated with 1∶1000 diluted secondary anti-rabbit antibody (BA-1000; Vector Laboratories, Burlingame, CA) for one hour at room temperature. Horseradish peroxidase conjugated streptavidin (SNN1004; Vector Laboratories, Burlingame, CA) at a dilution of 1∶1000 was added to the slides and incubated for 30 min. Immunoreactivity was detected using DAB (SK-4100; Vector Laboratories, Burlingame, CA). Next, the nuclei were stained with hematoxylin (Biocare Medical, Concord, CA) for 30 sec. The slides were subsequently washed in water and increasing gradients of ethanol, then placed in xylene before mounting in a xylene-based mounting solution (Fisher Scientific Inc, Hanover park, IL). The immunostained sections were observed under a microscope.
4
Immunohistochemistry of Nodal in DTIC-Resistant Melanoma
5
Immunohistochemical Detection of Salmonella
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Paraffin-embedded tissue samples were sectioned at
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl2) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl2) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.
6
Immunohistochemical Analysis of Cytokines
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DIVA antigen retrieval solution, Background Sniper blocking agent, Universal negative, Mach 4 HRP antibodies, DAB Peroxidase, and Hematoxylin were all purchased from Biocare Medical. IL-10 and TGF-β antibodies were purchased from Thermo Fisher Scientific. GAPDH polyclonal antibody was purchased from cell signaling.
7
Immunohistochemistry Reagents and Antibodies
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DIVA Decloaker 10x, Background Sniper, Mach 4 HRP polymer, DAB Peroxidase, Hematoxylin, Hydrophobic pen, and Universal Negative antibodies were all obtained from Biocare Medical, Concord, CA. Anti-JAG2 (LifeSpan Biosciences, Inc. Seattle, USA), Anti-AURKA (Sigma-Aldrich, St. Louis, USA), and anti-PGK1 (Abcam, Cambridge, UK) were stored at − 20 °C. Anti-HPRT monoclonal antibody (Abcam, Cambridge, UK) was aliquoted and stored at − 20 °C. GAPDH polyclonal antibody (Cell signaling) was aliquoted and stored at − 20 °C. Tween20 (Fisher Reagents, Waltham MA) was stored at room temperature. Hydrogen Peroxide at 30% (Fisher Reagents, Waltham MA) was stored at 4 °C.
8
Immunohistochemical Staining of Tie-2 Protein
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Sections rehydrated with standard procedures were incubated with 3% hydrogen peroxide (Sigma-Aldrich) for 10 minutes at room temperature. Antigen retrieval was performed with sodium citrate buffer at pH 6 in a pressure cooker for 10 minutes. Sections were then blocked with normal goat serum diluted in TBS for 1 hour. After the blocking, the sections were incubated with antibody against Tie-2 (1: 5000) (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour, followed by a 1 hour incubation with biotinylated rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) and a 30 minute incubation with VECTASTAIN® ABC Reagent complex. Signals were developed with the ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories). Slides were then counterstained with hematoxylin (Biocare Medical, Concord, CA, USA) before being mounted for analysis under the microscope.
9
Vaginal Cytology for Rat Estrous Cycle
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Daily, vaginal smears were stained to determine the cycle phases of the rats. The slides with the smears were first stained with a ready-to-use hematoxylin (Biocare Medical, CA, USA) for 10 min and gently washed with tap water. Slides were dipped in a saturated lithium carbonate solution for 3 min to intensify the staining, washed with water to remove the salt, and air dried at room temperature (RT). Then, the slides were covered with alcoholic eosin (Biocare Medical, CA, USA) for 10 min, washed with 70% ethanol, and air dried at RT. The vaginal smears were observed under an optical microscope Olympus BX41 (Olympus, PA, USA).
10
Immunohistochemical Identification of Immune Cell Subsets in Breast Cancer Sentinel Lymph Nodes
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Archived or fresh formalin-fixed paraffin-embedded biopsies of SLNs from breast cancer patients were sectioned and affixed to microscope slides. They were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water. Antigen retrieval was performed in a Digital Decloaking Chamber in DIVA Decloaker solution (Biocare Medical, Concord, California, USA). Tissue was stained with the following purified primary antibodies: mouse anti-human Cytokeratin (clone AE1/AE3; Biocare Medical); mouse anti-human CD20 (clone L26; Biocare Medical); rabbit anti-human CD3 (clone SP7; Biocare Medical), and mouse anti-human CD1a (clone CD1a007; Biocare Medical). Slides were subsequently stained with IgG secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase polymers. The antibody complexes were developed with diaminobenzidine (Biocare Medical), fast red (Biocare Medical), fast blue (Biocare Medical), NBT-BCIP (DAKO, Carpinteria, CA, USA), or VIP (Vector Laboratories, Burlingame, CA, USA). The cell nucleus was stained with hematoxylin (Biocare Medical).
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