Histofine simple stain max po m

Formalin-fixed brains were immersed in 98% formic acid to reduce infectivity and then embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin for evaluation of neuropathological changes. After epitope retrieval, PrP^Sc immunocytochemistry was performed using the mAbs T1 [14 (link)] or SAF84 (Bertin Pharma, Montigny le Bretonneux, France) [15 (link)]. Immunoreactions were developed using the anti-mouse universal immunoperoxidase polymer [Nichirei Histofine Simple Stain MAX-PO (M); Nichirei, Tokyo, Japan] as the secondary antibody and were visualized with 3,3′-diaminobenzedine tetrachloride as the chromogen.

The deparaffinized sections were digested with target retrieval solution (Dako North America, Inc., Carpinteria, CA) for 20 min, and treated with 3% hydrogen peroxide (WAKO Pure Chemical Industries, Ltd., Osaka, Japan) to block endogenous peroxidase activity. The sections were incubated overnight at 4 °C with a Rad goat polyclonal (1:100 dilution), or Rem1 mouse monoclonal antibody (1:200) (Santa Cruz Biotechnology, TX), and subsequently treated with peroxidase-labeled anti-goat (Histofine Simplestain max PO (G), Nichirei Bioscience, Tokyo, Japan) and anti-rabbit immunoglobulin (Histofine Simplestain max PO (M), Nichirei Bioscience) at 25 °C for 60 min. The signal was developed using peroxidase substrate 3-amino-9-ethylcarbazole (Histofine Simplestain AEC Solution, Nichirei Bioscience), which yielded a brown reaction product. The sections were counterstained with methyl green and examined under the microscope.

After appropriate epitope retrieval by hydrate autoclaving in 10 mM citrate buffer (pH 6.0) at 121°C for 3 min or with a combination of enzymatic and chemical treatments [32 (link)], sections were incubated with mAbs 3F4 or SAF84. An indirect horseradish peroxidase polymer system (Histofine Simple Stain MAX-PO [M]; Nichirei, Tokyo, Japan) with 3, 3'-diaminobenzedine tetrachloride as the chromogen was used for visualization. Sections were lightly counterstained with Mayer’s hematoxylin.

After appropriate epitope retrieval with either hydrate autoclaving or a combination of enzymatic and chemical treatment, IHC was carried out using the monoclonal antibodies (mAbs) 2G11, 12F10, or SAF84 (SPI-Bio, Montigny le Bretonneux, France) followed by an anti-mouse, universal horseradish peroxidase (HRP)-conjugated polymer (Nichirei Histofine Simple Stain MAX-PO (M); Nichirei Biosciences Inc., Tokyo, Japan) as the secondary antibody, and visualized with 3,3′-diaminobenzedine tetrachloride as the chromogen, as previously described [20 (link)]. Finally, the sections were lightly counterstained with Mayer’s hematoxylin.

Hearts were fixed in 4% (wt/vol.) paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with Sirius Red F3BA (0.5% wt/vol. in saturated aqueous picric acid; Aldrich Chemical Company, St Louis, MO, USA) for the measurement of collagen volume fraction. Cardiac interstitial fibrosis and the ratio of wall to lumen area of coronary artery were quantified, as described [34 (link)]. The positive area of fibrosis per field area was assessed by examining at least 10 fields per rat using Lumina Vision version 2.2 analysis software.
For ED-1 immunohistochemistry, cardiac sections were incubated overnight with ED1 antibody (×100; BMA Biomedicals, Switzerland) followed by Histofine simple stain Max-Po (M) (Nichirei, Biosciences, Tokyo, Japan). The number of cardiac ED-1-positive cells per mm² was counted in a blinded manner by examining more than 10 fields per section using a microscope with X200 magnification. The average ED-1-positive cell number was obtained in each rat.