Heart tissue was fixed in 10% buffered formalin, embedded in paraffin, and cut into 5-µm-thick sections. The sections were stained with Masson’s trichrome to determine the percentage of the infarct size, and the morphology of the infarcted size was identified using ImageJ software (ver. 1.47v; U.S. National Institutes of Health, Bethesda, MD). The percentage of the infarct size was assessed as the total infarct circumference divided by the total LV circumference × 100, as described [16 ]. Immunohistochemistry staining was performed by the immune-peroxidase method using paraffin-embedded tissue sections. After inhibition of endogenous peroxidase activity, the sections were incubated with primary rat anti-mouse Mac-3 antibody (#550292, BD Bioscience, Franklin Lakes, NJ), or rabbit anti-human CD3 antibody (#20006172F, Dako Autostainer/Autostainer Plus, Dako, Glostrup, Denmark) and then incubated at 4°C overnight with the respective secondary antibodies. Following visualization with 3,3′-diaminobenzidine (#10107863, Sigma–Aldrich, St. Louis, MO) according to the manufacturer’s instructions, the sections were finally counterstained with Mayer’s hematoxylin (Wako, Tokyo). All images were digitized using a microscope (Olympus AX80; Olympus Optical, Tokyo) equipped with a high-resolution camera (Nikon D2X; Nikon, Tokyo).
Dako autostainer autostainer plus
Manufactured by Agilent Technologies
Sourced in Italy
The Dako Autostainer/Autostainer Plus is a fully automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining instrument. It is designed to perform standardized and reproducible staining of tissue sections on glass slides.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Mayer s hematoxylin, by Fujifilm (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Envision flex mini kit, by Agilent Technologies (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Envision flex peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Envision flex substrate buffer, by Agilent Technologies (1 mentions)
Envision flex hematoxylin, by Agilent Technologies (1 mentions)
3 protocols using dako autostainer autostainer plus
1
Quantifying Myocardial Infarct Size
2
Immunohistochemistry Protocol for Breast Cancer Biomarkers
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The immunohistochemistry technique was performed using the EnVision FLEX Mini Kit, High pH high‐sensitivity visualisation system (Dako Autostainer/Autostainer Plus, Dako). The PT Link (Dako) module was used to pretreat the samples at a maximum temperature of 95°C with the corresponding retrieval solution according to the antibody used.
The sections were incubated with the primary antibodies:
The sections were incubated with the primary antibodies:
IO Path Mouse Anti‐Human Progesterone Receptor Monoclonal Antibody. Clone PR10A9. Prediluted (Beckman Coulter).
Mouse Anti‐Human Oestrogen Receptor α Monoclonal Antibody. Clone 1D5. Dilution 1:35 (Dako).
Rabbit Anti‐Human HER2 Polyclonal Antibody. Dilution 1:250 (Dako).
Mouse Anti‐Human COX‐2 Monoclonal Antibody. Clone CX‐294. Dilution 1:100 (Dako).
3
Immunostaining of CD1a-Positive Structures
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After de-paraffinization, 5 μm thick sections were immunostained to visualize CD1a-positive structures using Dako Autostainer/Autostainer Plus (Dako, Milan, Italy) and CD1a antibodies (M3571 anti-human CD1a Dako) diluted 1:100 in EnVision buffer. Each section was incubated with peroxidase-blocking serum (EnVision FLEX Peroxidase-Blocking Reagent, Dako) for 5 minutes to quench non-specific binding and then for 30 minutes with the primary antibody. After this, sections were incubated with a labeled polymer (EnVision FLEX /HRP, Dako) for 20 minutes and 3,3’-diaminobenzidine (EnVision FLEX Substrate buffer, + DAB + Chromogen; Dako) to label positive primary antibody binding. All the sections were finally counterstained with Hematoxylin (EnVision FLEX Hematoxylin, Dako) for 5 minutes to reveal the presence of nuclei, then dehydrated with an increasing scale of alcohol solutions (70%, 95%, 99%), cleared with xylene and mounted. These steps were performed at room temperature.[16] (link)
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