The liver and perigonadal adipose tissues were fixed with 10% neutral buffered formalin. After deparaffinization and rehydration, paraffin sections with 4-μm thickness were stained with H&E and Sirius Red. TUNEL staining was performed with an ApopTag Peroxidase In Situ Apoptosis Detection Kit (S7101; Merck-Millipore). Paraffin-embedded liver and adipose tissues were subjected to immunohistochemistry analyses. Endogenous peroxidases were blocked with 3% H2O2 for 30 minutes and nonspecific binding was blocked further with 3% normal goat serum for 1 hour. Tissues were incubated with primary antibody overnight at 4°C and incubated with the ABC staining kit (PK-4001, rabbit IgG; Vector Laboratories, Inc, Burlingame, CA). Reactions were developed with 3,3′-diaminobenzidine tetra hydrochloride peroxidase substrate (SK-4105; Vector Laboratories, Inc), according to the manufacturer’s instructions, and then slides were counterstained with hematoxylin.
Sk 4105
Manufactured by Vector Laboratories
Sourced in United States, Canada
SK-4105 is a laboratory equipment used for sample preparation. It is designed to facilitate the homogenization and disruption of various biological samples, such as tissues, cells, and microorganisms, to release their contents for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
H 3300, by Vector Laboratories (5 mentions)
Anti rabbit igg polymer detection kit, by Vector Laboratories (5 mentions)
S 2012, by Vector Laboratories (4 mentions)
Dako autostainer, by Agilent Technologies (4 mentions)
Pk 6100, by Vector Laboratories (3 mentions)
Vector antigen retrieval solution, by Vector Laboratories (3 mentions)
Aperio at2 slide scanner, by Leica camera (2 mentions)
Bp 9100, by Vector Laboratories (2 mentions)
Mp 7500, by Vector Laboratories (2 mentions)
Immpact dab, by Vector Laboratories (2 mentions)
Ba 1000, by Vector Laboratories (2 mentions)
Bloxall, by Vector Laboratories (2 mentions)
Mp 7401, by Vector Laboratories (2 mentions)
H 3401, by Vector Laboratories (2 mentions)
Ba 1100, by Vector Laboratories (2 mentions)
Armc5 antibody, by Novus Biologicals (2 mentions)
Histoclear, by National Diagnostics (2 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Bouin s solution, by Merck Group (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
45 protocols using sk 4105
1
Histological Analysis of Liver and Adipose Tissue
2
Quantification of p-HP1γ in Xenograft Tissues
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After sacrifice, formalin-fixed paraffin embedded xenograft tissues were stained for p-HP1γ. Tissues were deparaffinized using the standard procedure and unmasking/antigen retrieval was performed using pH 6.0 solution for 20 min at 98 °C in water bath. Tissue sections were stained for p-HP1γ using primary antibody Phospho-HP1γ (Ser83) (Cat. No: 2600, Cell Signaling technologies, 1:200) and secondary antibody Biotinylated antibody Anti-Rabbit (BP-9100, Vector Laboratories, 1:200). This was followed by Vectastain ABC solution incubation (PK-6100, Vector laboratories, 1:150) and DAB staining (SK-4105, Vector laboratories) as per the manufacturer’s protocol. Stained slides were scanned using Leica Aperio AT2 slide scanner. The criteria for senescent staining used for quantification was a very prominent nuclear staining in which the nucleus was bigger in size and its staining was darker brown than the other cells.
3
Immunohistochemical Analysis of Testicular Tissues
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Testes were fixed in Bouin’s solution (Sigma-Aldrich) overnight and then embedded in paraffin. Sections (5 μm) were mounted on poly-L-lysine-coated glass slides, deparaffinized, and rehydrated. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) at 120 °C for 10 min. The slides were incubated for 10 min in 3% H2O2 in methanol to block endogenous peroxidase activity. Then, the samples were blocked using 5% goat serum for 30 min. The slides were incubated in rabbit polyclonal anti-ESR1 antibody (1:200) and goat polyclonal anti-HSD3B antibody (1:500) overnight at 4 °C. Normal rabbit IgG and goat IgG were used as negative controls, and antigen absorption tests were performed to determine the specificity of the polyclonal antibodies (Supplementary Fig. S1 ). After washing in PBS, the slides were incubated in peroxidase-labeled goat anti-rabbit IgG (1:200) or rabbit anti-goat IgG (1:500) for 30 min. After washing in PBS, the signal was developed using a 3,3′-diaminobenzidine (DAB) substrate kit (SK-4105, Vector Laboratories, Burlingame, CA, USA), and nuclei were stained using Harris hematoxylin. Permanently mounted slides were observed and photographed using a microscope (IX71, Olympus, Tokyo, Japan) equipped with a digital imaging system (DP71, Olympus).
4
c-FOS Immunohistochemistry in P2rx7 Mice
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Two slices from three saline- and four PCP-treated animals per genotype (P2rx7+/+ and P2rx7−/−) were incubated for 10 min in 3% H2O2 in PB, rinsed three times for 10 min in PB, and three times for 10 min in PB. The pre-made blocking buffer (ImmPRESS UNIVERSAL REAGENT, Vector Laboratories, MP-7500) was supplemented with 0.5% Triton X-100 (Tx, Merck-Sigma), and 7.5% of normal donkey serum (NDS, Jackson Immunoresearch, Europe). After 1 h of blocking, slices were incubated with the primary rabbit anti-c-FOS antibody (1:1,000, Santa Cruz Biotechnology sc-52, Dallas, TX, USA) in PB containing 0.05% sodium azide (Merck-Sigma) for 24 h at room temperature (RT) and 72 h at 4°C. Eventually, the slices were rinsed in PB and incubated with the premade secondary anti-mouse/rabbit HRP-conjugated ImmPRESS UNIVERSAL REAGENT (Vector Laboratories, MP-7500) for 1 h. DAB was developed using the commercially available DAB-REACTION KIT (Vector Laboratories, SK-4105) according to the manufacturer’s instructions.
5
TREM Antibody Expression in Colon Tissue
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A human colon tissue array (Product No BC051110) was purchased from U.S. Biomax Inc. (Rockville, MD, USA), and the analysis was conducted according to the manufacturer’s suggested protocol. The tissue array included 10 normal tissues (J3–J12) and 110 cancer tissues. The slide was baked at 60 °C for 2 h, deparaffinized and rehydrated. Antigens were then retrieved by submerging the slide in boiling sodium citrate buffer (10 mM, pH 6.0) for 10 min. The sample was blocked with 3% BSA in 1X PBS/0.03% Triton X-100 in a humidified chamber for 1 h at room temperature and then incubated with a TREM antibody (reconstituted at 0.2 mg/mL in 1× PBS/0.03% Triton X-100) at 4 °C in a humidified chamber overnight. The slide was washed, hybridized with a secondary antibody (anti-goat, chicken antibody) conjugated with horseradish peroxidase (HRP, 1:1000) for 1.5 h at room temperature in the dark. The protein levels were visualized by colorimetric development of HRP using ImmPACT™ DAB with a metal enhancer (Vector Labs #SK-4105). The tissues were observed under a light microscope (X200).
6
Immunohistochemistry on Paraffin Sections
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A total of 5 µM thick paraffin embedded tissue sections were dewaxed and rehydrated for immunohistochemistry. Antigen retrieval were performed by boiling in the Antigen Unmasking Solution (H-3300, Vector laboratories) at 70% power for 3.5 min, and 20% power for 12 min using microwave. The slides were permeabilized by 0.2% Triton-100 for 10 min, and blocked by 3% hydrogen peroxide in methanol for 10 min and 5% normal donkey serum for 1 h at room temperature. Then they were incubated with specific primary antibody at 4 °C overnight (Supplementary data 6 ).
On the second day, the slides were incubated with biotinylated secondary antibody () for 1 h at room temperature, followed by the ABC reagent (PK-6100, Vector laboratories) for 1 h at room temperature. Signal was developed by DAB (SK-4105, Vector laboratories) for 30 s. The slides were counterstained with hematoxylin, dehydrated, cleared and mounted on Permount medium. The images were taken using Axiocam microscope camera (Zeiss).
On the second day, the slides were incubated with biotinylated secondary antibody () for 1 h at room temperature, followed by the ABC reagent (PK-6100, Vector laboratories) for 1 h at room temperature. Signal was developed by DAB (SK-4105, Vector laboratories) for 30 s. The slides were counterstained with hematoxylin, dehydrated, cleared and mounted on Permount medium. The images were taken using Axiocam microscope camera (Zeiss).
7
Immunohistochemical Analysis of Ucp1 in Adipose Tissue
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Adipose tissues were fixed in 10% formalin for 24 h at room temperature. Then, the tissues were embedded into paraffin and cut into 4 μm thick slices, deparaffinized, and rehydrated using xylene, ethanol, and water by standard methods. Immunohistochemistry was performed on a Dako Autostainer (Dako). Slides were incubated with 3% hydrogen peroxide and 2.5% normal horse serum (S-2012, Vector), followed by incubation with rabbit polyclonal anti-Ucp1 primary antibody diluted 1:200 in 2.5% normal horse serum (S-2012, Vector) for 60 min. Signals were detected with an anti-rabbit IgG Polymer Detection Kit (MP-7401, Vector). Labeling was visualized with 3,3′-diaminobenzidine (DAB) as the chromogen (SK-4105, Vector). Slides were counterstained with Harris hematoxylin (EK Industries), and whole-slide digital images were collected with an Aperio Scan Scope slide scanner (Aperio).
8
Immunohistochemical Analysis of HNF4α in Cirrhotic Livers
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Paraffin‐embedded liver tissue was deparaffinized with xylenes and dehydrated with ethanol. Antigen unmasking was performed by boiling in citrate buffer at pH = 6.0. The slides were then incubated in 3% hydrogen peroxide, blocked with normal animal serum, and subsequently left incubating overnight at 4°C with primary antibodies. The primary antibodies used are listed in Supporting Table S1 . Tissue sections were then incubated with biotinylated secondary antibody corresponding to the animal species of the primary antibody (BA‐1000; Vector Laboratories, Burlingame, CA) and exposed to 3,3′‐diaminobenzidine (SK‐4105; Vector Laboratories) to visualize the peroxidase activity. Counterstaining was performed with Richard‐Allan Scientific Signature Series Hematoxylin (Thermo Fisher Scientific). For quantification, immunoreactivities of nuclear and cytoplasmic HNF4α were independently graded by 2 liver pathologists, with 1,000 hepatocytes in three high‐power fields being counted per sample. Normal livers (n = 4), Child‐Pugh B (n = 5), and Child‐Pugh C (n = 2) were included for these analyses. The Child‐Pugh B and C livers were grouped as cirrhotic human liver, and the results are expressed as percentage over the total number of cells counted.
9
Quantifying Fibrin/Fibrinogen in Airway Casts
10
Quantifying Neuronal Death in Drosophila Brains
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To measure neuronal death in Drosophila brains, we used a commercially available DNA fragmentation detection kit for TUNEL staining (Calbiochem, TdT FragEL) using 4 μm sections of formalin-fixed, paraffin embedded Drosophila brain tissue. As directed in the provided protocol, DAB (Vector Laboratories, SK-4105) was used for detection of biotin-labelled deoxynucleotides at exposed ends of DNA fragments. Brightfield microscopy (Nikon Eclipse Ci-L) was used to quantify TUNEL-positive cells throughout the Drosophila brain.
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