For histological analyses, liver samples (the same lobe from each mouse) were fixed overnight in 10 per cent neutral buffered formalin, dehydrated in a graded alcohol series, and embedded in paraffin; 4-µm sections were prepared. Epitope retrieval was performed by autoclave pretreatment in Histofine® antigen retrieval solution (pH 9) (Nichirei, Tokyo, Japan). Endogenous peroxidase activity was blocked with 3 per cent hydrogen peroxide, and the sections were incubated with diluted 1 : 15 antibromodeoxyuridine peroxidase Fab fragments overnight at 4°C. The sections were also incubated with 1 : 100 Phospho-Smad2 (138D4; Cell Signaling Technology, Danvers, Massachusetts, USA) and 1 : 100 TSP-1 (A6.1; Thermo Scientific, Waltham, Massachusetts, USA) antibodies. Diaminobenzidine solution was used as chromogen, followed by counterstaining with Mayer's haematoxylin.
Histofine antigen retrieval solution ph 9
Manufactured by Nichirei Biosciences
Sourced in Japan
Histofine® antigen retrieval solution (pH 9) is a laboratory reagent designed to facilitate the retrieval of antigenic sites in formalin-fixed, paraffin-embedded tissue sections prior to immunohistochemical staining. The solution has a pH of 9.
Automatically generated - may contain errors
Lab products found in correlation
Envision plus, by Agilent Technologies (1 mentions)
Envision kit hrp dab, by Agilent Technologies (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Histofine max po kit, by Nichirei Biosciences (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Picrosirius red stain kit, by Polysciences (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 conjugated donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Donkey serum, by Merck Group (1 mentions)
A31573, by Thermo Fisher Scientific (1 mentions)
Ab5084p, by Merck Group (1 mentions)
A21450, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
5 protocols using histofine antigen retrieval solution ph 9
1
Histological Analysis of Liver Samples
2
Immunohistochemical Analysis of CRC Samples
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Formalin fixed, paraffin-embedded blocks of CRC resected specimens were sliced to 3-µm sections. Sections were autoclave-pretreated in histofine antigen retrieval solution (pH9) (Nichirei, Tokyo, Japan). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, and the sections were incubated with 50 times diluted primary antibodies (EZH2 and p27) over night at 4°C. A subsequent reaction was performed with a biotin-free horseradish peroxidase enzyme-labeled polymer of the Envision Plus detection system (Dako, Glostrup, Denmark). The reaction was visualized with a diaminobenzidine solution, followed by counterstaining with Mayer’s hematoxylin.
3
Paraffin Embedding and Immunostaining of 293T Cells
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For preparation of the paraffin block of 293T cells, the cells were fixed in 20% of formalin/PBS for 24 h. After removing the formalin, alcohol dehydration and paraffin permeation were done using Tissue-Tek VIP5Jr (Sakura, Alphen aan den Rijn, The Netherlands). Paraffin blocks were sectioned at 3-μm thickness. The sections were then transferred to coating slide glasses (Muto pure chemicals, Bunkyo-ku, Tokyo, Japan). After paraffin removal, the paraffin sections of the 293T and ATL cells were treated with 3% H2O2. Antigen-retrieval treatment was done using Histofine antigen retrieval solution pH9 (Nichirei, Chuo-Ku, Tokyo, Japan) for 20 min under microwave radiation. After reaction with the first antibody, anti-EVC antibody (HPA008703, 1:400; SIGMA), and the second antibody (K5027, ENVISION Kit/HRP [DAB]; Dako, Bunkyo-ku, Tokyo, Japan), the sections were colored using ENVISION Kit/HRP [DAB] DAB+ (K3468; DAKO). Finally, the sections were stained with hematoxylin.
4
Isolation and Characterization of Cells
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Uranyl acetate (UA) dihydrate (purity >98.0%) was purchased from Fluka (Buchs, Switzerland). Collagenase type II was from Worthington Biochemical Corp. (Lakewood, NJ). Percoll was purchased from GE Healthcare UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Trypan blue solution, propidium iodide, Hoechst 33342, and pyronin Y were purchased from Sigma‐Aldrich Co. (St. Louis, MO). Hank's balanced salt solution (HBSS) was from Invitrogen (Carlsbad, CA). Can Get Signal® solution B was from Toyobo Life Science Department (Osaka, Japan). Citrate buffer solution was from Mitsubishi Chemical Medience (Tokyo, Japan). Histofine Antigen Retrieval Solution pH9® and Histofine MAX PO kit were from Nichirei Bioscience (Tokyo, Japan). ApopTag® Plus In Situ Apoptosis Detection Kit was from Chemicon‐Millipore (Temecula, CA). Picrosirius red stain kit was from Polysciences, Inc. (Warrington, PA). The antibodies listed in Table 1 were used as primary antibodies. Alexa Fluor® 633‐conjugated donkey anti‐goat IgG (Invitrogen), Alexa Fluor® 546‐conjugated goat anti‐rabbit IgG (Invitrogen), Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (Invitrogen), and Histofine Simple Stain Max PO (Nichirei Bioscience) were used as secondary antibodies.
5
Immunofluorescence Labeling of Olfactory Bulb
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Mice were deeply anesthetized with an overdosing i.p. injection of pentobarbital. After intracardiac perfusion with 4% PFA (Nacalai Tesque, #26126-25) in PBS, the OB was dissected and post-fixed in 4% PFA in PBS overnight. The OB was then cryoprotected with 30% sucrose and then embedded in OCT Compound (Sakura, #4583). Frozen sections were cut at 18 mm thick with a cryostat (Leica).
Antigen retrieval was performed by heating in a microwave oven for 5 min in Histofine antigen retrieval solution pH 9 (Nichirei, #415201). Sections were pretreated with 4% PFA in PBS and 5% Donkey serum (Sigma, #D9663) in PBS with 0.1% Triton X-100. Rabbit anti-Drd2 (Millipore, AB5084P, RRID: AB_2094980) and guinea pig anti-Gabbr1 (Millipore, AB2256, RRID: AB_11210385) were used at 1:100 and 1:500 dilutions, respectively. AlexaFluor647-conjugated secondary antibodies (Thermo Fisher Scientific, A31573 and A21450, RRID: AB_2536183 and AB_141882) were used at 1:200 dilutions. Sections were counterstained with DAPI (Dojindo, #D523). Immunofluorescence was imaged with an inverted confocal laser-scanning microscope (Olympus, FV1000) using a 20x dry objective lens (Olympus).
Antigen retrieval was performed by heating in a microwave oven for 5 min in Histofine antigen retrieval solution pH 9 (Nichirei, #415201). Sections were pretreated with 4% PFA in PBS and 5% Donkey serum (Sigma, #D9663) in PBS with 0.1% Triton X-100. Rabbit anti-Drd2 (Millipore, AB5084P, RRID: AB_2094980) and guinea pig anti-Gabbr1 (Millipore, AB2256, RRID: AB_11210385) were used at 1:100 and 1:500 dilutions, respectively. AlexaFluor647-conjugated secondary antibodies (Thermo Fisher Scientific, A31573 and A21450, RRID: AB_2536183 and AB_141882) were used at 1:200 dilutions. Sections were counterstained with DAPI (Dojindo, #D523). Immunofluorescence was imaged with an inverted confocal laser-scanning microscope (Olympus, FV1000) using a 20x dry objective lens (Olympus).
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