Histofine sab po r kit

For light microscopic analysis, tumor tissues were fixed with 3% formaldehyde, paraffin embedded, and stained with hematoxylin and eosin (H&E) using standard techniques. Antibodies used were mouse CD31 (1:100, Cell Signaling, 77699), PDGFRB (1:100, R & D Systems, BAF1042), RAB27A (1:100, Cell Signaling, 69295), SYTL2 (1:100, Santa Cruz Biotechnology, sc393847), VWF (1:100, Santa Cruz Biotechnology, sc365712), human CD31 (1:100, Abcam, ab28346), FLAG-tag (1:100, Sigma-Aldrich, F3165), aSMA (1:100, DAKO, MO851), TFE3 (1:100, Santa Cruz Biotechnology, sc5958), and NG2 (1:100, Millipore, AB5320). Heat-mediated antigen retrieval was performed in Tris-EDTA buffer at pH 6.0. Immunohistochemical staining was performed using the Simple Stain MAX-PO kit (Nichirei Bioscience), the Histofine SAB-PO (R) kit (Nichirei Bioscience).

An immunohistochemical examination was carried out to analyze the biological characteristics of the cells in the dental pulp tissues cultured using the improved CMDPT. The sections were deparaffinized with xylene, rehydrated in descending concentrations of ethanol, and washed in PBS. After antigen retrieval in a 0.01 mol/L sodium-citrate-buffered solution (pH 6.0) at 98 °C for 45 min, the sections were cooled and washed well with PBS. Endogenous peroxidase was blocked by treatment with 3% H₂O₂ in methanol for 1 h at room temperature (RT). After treatment with 10% normal goat serum at RT for 10 min, the sections were incubated with rabbit anti-Ki-67 antibody (Abcam, Cambridge, UK) (diluted 1:200 in PBS containing 1% bovine serum albumin) or rabbit anti-stromal cell-derived factor-1(SDF1) antibody (Abcam, Cambridge, UK) (diluted 1:1000 in PBS containing 1% bovine serum albumin) at 4 °C overnight. After washing with PBS, the localization of each molecule was determined using a Histofine SAB-PO (R) kit (Nichirei Biosciences, Inc., Tokyo, Japan) and 3, 3′-diaminobenzidine (DAB) substrate kit (Nichirei Biosciences, Inc.). The sections were counterstained with hematoxylin and mounted. The specificity of the immunoreaction was confirmed by incubation with normal rabbit IgG instead of primary antibody.

Paraffin-embedded ACLs were sectioned longitudinally (5 μm thick). In order to perform the staining procedure, first these sections were deparaffinized, rehydrated, and then antigen retrieval was accomplished by 10 mM citrate buffer at 60 °C for 1 h. These sections were incubated with rabbit primary antibodies against MMP-1 (1:100), MMP-3 (1:50), or IL-6 (1:400) at 4 °C overnight, rinsed with phosphate-buffered saline, and incubated with reagents included in the Histofine SAB-PO(R) kit (Nichirei Biosciences Inc., Tokyo, Japan). All sections were visualized using the Olympus BX53 microscope (Olympus, Tokyo, Japan); images were captured digitally using the Leica DFC7000 T digital camera and processed using the Leica Application Suite Ver4.6 imaging software. The primary antibodies used were rabbit anti-MMP1 antibody (1:100, ab52631, lot: GR3261996-3; Abcam, Cambridge, UK), rabbit anti-MMP3 antibody (1:50, ab52915, lot: GR3249690-2; Abcam), and rabbit anti-IL-6 antibody (1:400, ab6672, lot: GR3195128-25; Abcam). The acquired images were analyzed using ImageJ Fiji (https://imagej.net/software/fiji/). Color deconvolution in ImageJ Fiji was used to separate the 3,3′-diaminobenzidine staining, and the mean gray values in the nucleus and cytoplasm were measured and quantified.

Immunohistochemistry was performed according to the method described in our previous report [44 (link)]. Briefly, an anti-YAP1 antibody (HPA070359; Sigma-Aldrich), a Histofine SAB-PO(R) kit, and 3, 3′-diaminobenzidine substrate-chromogen (Nichirei, Tokyo, Japan) were used.

Histological diagnosis was performed with all the tissue samples obtained by surgical resection and finally classified into PMCA and DPAM. Presence of signet ring cells was judged positive if the cells were observed at any percentages in the tumorous components but not in isolated mucin pools. Tissue sections of the 18 tumors were further analyzed by immunohistochemical staining with anti‐TP53 antibody (sc‐126, Santa Cruz Biotechnology, Inc., Delaware, CA), Histofine SAB‐PO(R) Kit (Nichirei, Tokyo, Japan), and ImmPACT DAB systems (VECTOR LABORATORIES, Burlingame, CA). Expression of TP53 was scored into three classes, namely normal, negative, and excessive, according to previous reports 6, 7. Normal staining referred to a weak focal nuclear staining, resembling a staining pattern for normal epithelium in less than 50% of tumor nuclei. A negative score indicated a complete lack of stained tumor nuclei (even though stromal cell nuclei showed normal pattern). The staining was classified as excessive if a strong nuclear staining was present in the majority of tumor cells (>50%).