5 µm formalin-fixed paraffin-embedded tumour sections were prepared, dewaxed, washed in down-graded alcohols and water. Antigen retrieval was conducted by immersing the sections in pre-heated 90–95°C Envision Flex Target Retrieval Solution of high pH (DAKO, USA) and microwaved for a further 15 to 30 min. Sections were allowed to cool down in the retrieval solution for at least 20 min at room temperature. Rabbit anti-αSMA antibody (Abcam, ab5694, dilution 1:500) was then applied to sections and incubated for 30 min at room temperature after blocking endogenous peroxidase. Detection was facilitated by the addition of EnVision FLEX/HRP (DAKO) and DAB chromogenic substrate (DAKO) for 30 min and 2 min, respectively, at room temperature. Haematoxylin was used for nuclear counterstain.
Envision flex target retrieval solution of high ph
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Envision Flex Target Retrieval Solution of high pH is a laboratory solution used in the process of antigen retrieval for immunohistochemistry analysis. It is designed to unmask or expose target antigens in formalin-fixed, paraffin-embedded tissue samples, facilitating the subsequent binding of specific antibodies for detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Ab5694, by Abcam (2 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Dab chromogenic substrate, by Agilent Technologies (1 mentions)
Genelute pcr clean up kit, by Merck Group (1 mentions)
Envision g 2 doublestain system, by Agilent Technologies (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions)
Opal 520 reagent, by Akoya Biosciences (1 mentions)
Opal 570 reagent, by Akoya Biosciences (1 mentions)
Vectra polaris imaging system, by PerkinElmer (1 mentions)
3 protocols using envision flex target retrieval solution of high ph
1
Immunohistochemical Staining of αSMA
2
Analyzing Cell Surface Glycoproteins
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The following materials were purchased from the manufacturers indicated: RNeasy Kit and RNase-Free DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and PowerSYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA, USA); GenElute PCR clean-up kit and 3,3′-diaminobenzidine from Sigma-Aldrich (St. Louis, MO, USA); and EnVision™ G|2 Doublestain System and EnVision FLEX Target Retrieval Solution of High pH from Dako (Glostrup, Denmark). All other chemicals were obtained from commercial sources and were of analytical grade.
The following antibodies were used in this study: rabbit anti-syndecan 2 (M-140) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); rabbit anti-glypican 1 polyclonal antibody from Thermo Scientific (Thermo Fisher Scientific Inc., Waltham, MA, USA); rabbit anti-glypican 5 monoclonal antibody from Novus Biologicals (Littleton, CO, USA); monoclonal anti-CS clone CS-56, purchased from Sigma-Aldrich (St. Louis, MO, USA); and anti-mouse (sc-2020) and anti-rabbit (sc-2004) secondary antibodies, from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The following antibodies were used in this study: rabbit anti-syndecan 2 (M-140) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); rabbit anti-glypican 1 polyclonal antibody from Thermo Scientific (Thermo Fisher Scientific Inc., Waltham, MA, USA); rabbit anti-glypican 5 monoclonal antibody from Novus Biologicals (Littleton, CO, USA); monoclonal anti-CS clone CS-56, purchased from Sigma-Aldrich (St. Louis, MO, USA); and anti-mouse (sc-2020) and anti-rabbit (sc-2004) secondary antibodies, from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
Multiplexed Immunostaining of Paraffin Sections
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After dewaxing and rehydration of the paraffin-embedded sections, the antigen retrieval was conducted using Envision Flex Target Retrieval Solution of high pH (DAKO). For FSTL1 and aSMA staining, the sections were blocked with Opal Antibody Diluent/Block (AKOYA), followed by incubation with FSTL1 (1:100, Abcam, ab71548) for 1 hour at room temperature. Signal was detected by incubating Opal Polymer horseradish peroxidase (HRP), Mouse plus Rabbit (AKOYA) for 30 minutes, followed by Opal 570 Reagent (AKOYA). Another round of antigen retrieval and antibody stripping were conducted before incubating with aSMA (1:100, Abcam, ab5694) for 1 hour at room temperature. Signal was detected by incubating Opal Polymer HRP, Mouse plus Rabbit (AKOYA) for 30 minutes, followed by Opal 520 Reagent (AKOYA). DAPI (AKOYA) was used to counterstain the nuclei. pAKT and pan-CK staining was done as described above, with pAKT (Ser473) (1:200, Cell Signaling Technology, 4060) and pan-CK (1:200, Abcam, ab7753) stained with Opal 570 and Opal 520 Reagent (AKOYA), respectively. The sections were imaged using Vectra Polaris imaging system (Perkin Elmer). Analysis to segment and quantify the cells based on their immunostaining were performed using inForm versions 2.2 (Akoya Biosciences).
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