TUNEL staining was performed to detect apoptotic cells as described previously [25 (link),26 (link)]. After being incubated in 20 μ g/ml proteinase K (Sigma), the serial sections used for HE staining were immersed in TDT buffer (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, and 1 mM cobalt chloride). TDT and biotinylated dUTP (both from Roche) were diluted with TDT buffer at concentrations of 0.15 e.u./ml and 0.8 nmol/ml, respectively. The solution was placed on the sections and then incubated at 37°C for 60 min. The sections were covered with streptavidin peroxidase (DAKO, Carpinteria, CA, USA) and stained with DAB as a substrate for peroxidase. Counterstaining was performed using Mayer’s hematoxylin.
Streptavidin peroxidase
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Streptavidin-peroxidase is a conjugate of streptavidin, a tetrameric protein, and the enzyme horseradish peroxidase. It is designed for use in various bioanalytical techniques that rely on the high affinity of streptavidin for biotin.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Leica dfc310 fx, by Leica camera (2 mentions)
Diaminobenzidine, by Agilent Technologies (2 mentions)
Eclipse te2000 u inverted microscope, by Nikon (2 mentions)
Diaminobenzidine substrate chromogen system kit, by BD (2 mentions)
Biotin blocking system, by Agilent Technologies (2 mentions)
Armenian hamster anti mouse cd54 icam 1, by Thermo Fisher Scientific (2 mentions)
Armenian hamster igg isotype control, by Thermo Fisher Scientific (2 mentions)
Biotin streptavidin conjugated goat anti armenian hamster igg h l, by Thermo Fisher Scientific (2 mentions)
Biotinylated dutp, by Roche (1 mentions)
Proteinase k, by Merck Group (1 mentions)
3 amino 9 ethylcarbazole, by Agilent Technologies (1 mentions)
Af 317 na, by R&D Systems (1 mentions)
Epitope retrieval solution, by Agilent Technologies (1 mentions)
Bond system, by Leica camera (1 mentions)
α sma, by Abcam (1 mentions)
Anti cytokeratin, by Abcam (1 mentions)
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd68, by Bio-Rad (1 mentions)
24 protocols using streptavidin peroxidase
1
Apoptosis Detection via TUNEL Staining
2
Immunohistochemical Analysis of FGF10 Expression
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We randomly chose 50 tissue specimens for FGF
staining according to higher and lower than the estimated
relative quantification (RQ) median (12.39) for FGF10.
The first paraffin separation was done at 55°C, then, after
20 minutes, three washes with xylene. Next, the samples
were rehydrated in ethanol concentrations of 100%, 95%,
and 80%. Subsequently, 3% hydrogen peroxide was
applied to block any endogenous peroxidase activity.
The prepared slides were heated in citrate acidic buffer
(pH=6.0) in an oven for 10 minutes through antigen
retrieval. Primary antibody (1:100) incubation at 4°C
was done for two hours, then each slide was incubated
for 30 minutes in biotin labelled with secondary antibody
(Abcam, ab80064, Cambridge, UK) and tracked with
incubation in streptavidin-peroxidase (Dakocytomation,
Inc., CA, USA). The Diaminobenzidine substrate was
added to the slides, and they were visualized under a
microscope. Finally, the cutting segments were stained
with haematoxylin and dehydrated. For checking the
negative control, the intended segment was incubated
with phosphate-buffered saline (PBS) instead of primary
antibody, followed by the rest of the previously mentioned
procedures. The positive control was brain-skin tissue
segments, which had high FGF10 expression (1 (link)).
staining according to higher and lower than the estimated
relative quantification (RQ) median (12.39) for FGF10.
The first paraffin separation was done at 55°C, then, after
20 minutes, three washes with xylene. Next, the samples
were rehydrated in ethanol concentrations of 100%, 95%,
and 80%. Subsequently, 3% hydrogen peroxide was
applied to block any endogenous peroxidase activity.
The prepared slides were heated in citrate acidic buffer
(pH=6.0) in an oven for 10 minutes through antigen
retrieval. Primary antibody (1:100) incubation at 4°C
was done for two hours, then each slide was incubated
for 30 minutes in biotin labelled with secondary antibody
(Abcam, ab80064, Cambridge, UK) and tracked with
incubation in streptavidin-peroxidase (Dakocytomation,
Inc., CA, USA). The Diaminobenzidine substrate was
added to the slides, and they were visualized under a
microscope. Finally, the cutting segments were stained
with haematoxylin and dehydrated. For checking the
negative control, the intended segment was incubated
with phosphate-buffered saline (PBS) instead of primary
antibody, followed by the rest of the previously mentioned
procedures. The positive control was brain-skin tissue
segments, which had high FGF10 expression (1 (link)).
3
Histochemical Analysis of DBA Lectin in Goat Endometrium
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To further confirm the incidence of DBA lectin positive uterine leukocytes in the non-pregnant goat endometrium, endometrium samples were prepared for histologic examination. Paraffin sections (7 µm) of endometrium were processed for DBA lectin cytochemistry according to Chen et al. [15 (link)]. Briefly, it consisted in incubated with biotinylated DBA lectin, followed by streptavidin-peroxidase (DAKO, CA) and revealed with 3,3,-diaminobenizidine (Sigma, St Louis, MO) and hydrogen peroxide reaction. The control reaction was performed by adding 0.1 M N-acetyl-D-galactosamine (NacGal) in the DBA lectin solution before the incubation with secretions.
4
Immunohistochemical Analysis of IL-17A
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5 µm sections of paraffin embedded skin samples were air-dried overnight at 37 °C, dewaxed and rehydrated. Stainings were performed by an automated BOND system (Leica) according to the manufacturer’s instructions: epitope retrieval was performed at pH6 in epitope retrieval solution (DAKO) and incubated with goat anti-human IL-17A (#AF-317-NA, R&D Systems) followed by a biotinylated anti-goat secondary antibody (#BA-9500-1.5, Vector Laboratories). For detection of specific binding, streptavidin peroxidase and its substrate 3-amino-9-ethyl-carbazole (DAKO) were used. All slides were counter stained with hematoxylin. Stainings without primary antibodies were used as negative control. Positive cells were counted in four to nine visual fields per condition.
5
Immunohistochemical Analysis of Cell Markers
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Cells were seeded on poly-L-lysine-coated glass coverslips in 6-well plates and received different treatments. After being fixed with 4% paraformaldehyde for 15 min, cells were permeabilized in 0.5% Triton X-100 for 20 min, and subsequently incubated with 0.3% hydrogen peroxidase for 10 min in order to block endogenous peroxidase activity. Slides were incubated with normal goat serum for 1 hour to block unspecific labeling. Subsequently, cells were incubated with the following antibodies overnight at 4°C: anticytokeratin (CST) and α-SMA (Abcam). Slides were then washed in phosphate buffer saline (PBS) followed by incubation with secondary antibody (Wuhan Boster Biological Engineering) for 1 hour at 37°C, followed by incubation with streptavidin-peroxidase (Dako) for 15 min at 37°C. 3,3′-diaminobenzidine (DAB, Dako) was applied as the chromogenic agent. Then slides were counterstained with hematoxylin, dehydrated in graded ethanol and coverslipped. Cells were observed under a fluorescent microscope.
6
Wound Healing Histological Analysis
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At the experimental endpoint at day 7, the entire wound was paraffin-embedded. At experimental endpoint at day 29, the wound area was cut into two halves. One half was paraffin embedded. The other half was used to measure the breaking strength. Paraffin-embedded sections (5 µm), n = 6 per group and time point, were deparaffinized and rehydrated. Antigen retrieval was performed in Tris-EDTA buffer containing 0.1% trypsin (Invitrogen, Carlsbad, CA). Endogenous peroxidase activity was quenched by exposing to 0.1% hydrogen peroxide in PBS containing 0.1% Tween 20 (PBST). After blocking with 4% nonfat milk powder in PBST, the sections were incubated with mouse anti-CD68 (1∶100; AbD Serotec, Düsseldorf, Germany) and goat anti-CD34 (1∶200; DakoCytomation, Glostrup, Denmark), respectively, followed by incubating with the corresponding biotinylated secondary antibodies (DakoCytomation). The antigen-antibody complex was detected by streptavidin-peroxidase (DakoCytomation) and 3,3′-diaminobenzidine (Sigma-Aldrich, Zwijndrecht, the Netherlands).
7
Immunohistochemical Evaluation of PPAR-γ and KIM-1
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Paraffin sections were subjected to alcohol and xylene gradient solutions, antigen retrieval, protein block, and incubation with primary antibodies against PPAR-γ polyclonal antibody (1:200, Abcam, MA, USA) and kidney injury molecule 1 (KIM-1) (1:500, rabbit IgG, Sino Biological, Beijing, China) overnight at 4 °C. After this time, the sections were incubated with streptavidin-peroxidase for 30 min (Dako, CA, USA). Periodic acid–Schiff staining was carried out according to the manufacturer’s instructions. The obtained microscope images were calculated using Leica DFC 310 FX image analysis software (Leica do Brasil Importação e Comércio Ltd., São Paulo, Brazil) and are expressed as the percentage/stained area.
8
Visualization of Viral RNA in Mouse Brain Tissue
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Brain tissues from uninfected age-matched, sex-matched mice were flash-frozen in liquid nitrogen. Tissue was cryosectioned at 10μm (Leica Cryostat CM1860). Sections were kept on ice and fixed for 10 minutes using Carnoy’s solution (25% v/v glacial acetic acid, 75% v/v absolute ethanol) and then rinsed for 10 minutes in distilled water. Tissues were incubated (5 min) with peroxidase block (Dako), and stained with the K1 anti-dsRNA antibody (English and Scientific Consulting, Hungary; 1:200 dilution in PBS, 30 minute incubation) that had been biotinylated using the Dako ARK peroxidase kit for mouse primary antibodies (Dako, K3955). Biotinylated-K1 antibody was detected using the streptavidin-peroxidase (30 minute incubation) and DAB+substrate-chromogen (3 minute incubation) reagents (Dako). Slides were photographed using an Olympus DP73 camera attached to an Olympus AX70 microscope. All of the images compared were obtained using the same instrument settings.
9
Immunohistochemical Analysis of Collagen Types I and II
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The slides were prepared as mentioned above. After deparaffinization, antigen retrieval process was performed by immersing the slides in boiling citrate buffer (pH 6) and cooled down at RT for 20 min. Next, sections were permeabilized in 0.2% Triton in PBS for 10 min at RT, washed three times with PBS, each for 5 min, and incubated in 4% hydrogen peroxide for 30 min at RT to block the endogenous peroxidase activity. Then, slides were incubated in hyaluronidase type II (Sigma) at a concentration of 1 mg/ml in PBS (pH 6) for 30 min at 37°C to break down the matrix and blocked in 10% FBS for 1 h at RT followed by incubating with primary antibodies anti-collagen type I (ab138492, Abcam) and anti-collagen type II (ab34712, Abcam) for 16 h at 4°C. After washing with PBS, the sections were incubated with anti-rabbit biotinylated antibody (1 : 500, Dako) for 30 min at RT, washed in PBS, incubated in streptavidin peroxidase (Dako) for 30 min at RT, then stained with 3,3′-diamino-benzidine (DAB, Dako) for 3 min, counterstained in Mayer's hematoxylin for 1 min, submerged in distilled water containing some drops of 1 M NH4OH (Sigma) for bluing, and mounted with DPX mounting medium. Photos were taken by using a light microscope (DM5000 B, Leica).
10
Histological Analysis of Cryo and FFPE Tumors
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5 μm and 3 μm sections of TissueTek-cryo-preserved and formalin-fixed paraffin-embedded (FFPE) tumors, respectively, were used for histological analyses. Hematoxylin and eosin staining (H&E, Sigma) was conducted on cryo and FFPE sections. Tumor grade was determined by an experienced pathologist in three tumors/group and 10 high-power fields each. TUNEL assay (Roche, Mannheim, Germany) was performed on cryo sections according to the manufacturer's instructions. For Ki-67 staining, cryo sections were fixed with formalin, peroxidase blocked (Dako, Hamburg, Germany), incubated for 1 hour at room temperature (RT) with biotinylated mouse anti-human Ki-67 (Dako), and visualized with streptavidin-peroxidase (Dako) and diaminobenzidine (Dako).
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