Vectastatin elite abc kit

The biopsy sections were subjected to IHC analysis to determine the expression of several cellular biomarkers following PEG treatment. These included EGFR, Ki-67, SNAIL, Cleaved Caspase-3 and E-cadherin, all of which have previously been reported by our group to be modulated by PEG in cell culture and animal models [14 (link)]. For these studies, 2–3 rectal biopsies were formalin fixed, paraffin embedded, sectioned and subjected to the IHC evaluation by standard techniques using appropriate primary antibodies [anti-Ki67 (1:250; AbCam, Cambridge, MA), anti-EGFR (1:200; Santa Cruz, CA, anti-Cleaved Caspase 3 (1: 100; Cell Signaling Technology, Danvers, MA)], anti-SNAIL (1:100; Snai1–T18; Santa Cruz, CA) and anti-E-cadherin (1:250; Cell Signaling Technology) followed by appropriate biotinylated secondary antibodies. The antigen-antibody complexes were detected with the Vectastatin Elite ABC kit (Vector Laboratories). For negative controls, sections were processed in the absence of the primary antibodies. IHC sections were scored by the study pathologist (CW), who was blinded to the study group. A semi- quantitative scale was used to evaluate immunoreactivity of epithelial cells. The extent of staining was graded and scored as 0 (negative staining); 1+ (10% stained cells), 2+ (10–50% stained cells), and 3+ (50% stained cells).

DRGs from segments L1–5 from both sides were excised separately, fixed at 4 °C in 4 % paraformaldehyde for 24 h, embedded in paraffin, cut into 5 μm sections, which were dewaxed and autoclaved for 15 min (120 °C, 1 bar). For CGRP labeling we applied overnight at 4 °C a mouse reactive anti-CGRP antibody (1:100; polyclonal against a synthetic rat Tyr-CGRP (23–37) conjugated to gamma globulin developed in goat; Cat. N^o BP022, Acris Antibodies, Herford, Germany). Sections were incubated for 2 h in biotinylated rabbit anti-goat antibody (1:200; DAKO, Glostrup, Denmark), then the avidin-biotin peroxidase complex (Vectastatin Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) was applied for 40 min. Sections were developed with Jenchrom px blue (JenLab, Jena, Germany). In control experiments the primary antibody was omitted or preincubated with 100 μg/ml CGRP (1–37) for 20 min (Bachem, Bubendorf, Germany).
The immunohistochemical labeling was evaluated as previously described [28 (link)]. In each second section the proportion of neuronal profiles with CGRP-like immunoreactivity (IR) was determined using a light microscope (Axioplan 2, Zeiss, Jena, Germany) coupled to an image analyzing system (AxioVision, Zeiss). For each mouse the average proportion of labeled neuronal profiles was calculated. At least 100 neuronal profiles with a visible nucleus per mouse were counted.

Mice (5 WTs and 5 Hets for POU3F2 staining, 7 WTs and 6 Hets for BCL11B staining, sex balanced, at the age of P60) were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. Whole brains were weighed and serially sectioned in the coronal plane at 75 μm using a vibratome and immunolabeled for either BCL11B (a marker for cortical layers V/VI) or POU3F2 (a marker for cortical layers II-IV). For each antibody, a set consisting of every eighth section was isolated and slide mounted. After drying overnight, antigen retrieval was performed by immersing in citrate buffer (pH 6.0) and pressure cooking for 10 min. The slides were then quenched in 3% hydrogen peroxide in absolute methanol for 10 min, immersed for 1 h in a blocking solution (2% bovine serum albumin, 0.2% dry milk, 0.8% Triton X-100 in PBS), and incubated overnight with a 1:500 dilution of either BCL11B (Abcam ab18465) or POU3F2 (Santa Cruz sc-393324). The next morning, BCL11B or POU3F2 incubated sections were reacted with appropriate biotinylated secondary antibody for 1 h (Sigma-Aldrich B7139; 1:200 or Vector Labs BA-9200; 1:200, respectively). The sections were then reacted with an avidin-biotin conjugate (ABC kit) for 1 h and visualized using the chromogen VIP (Vectastatin Elite ABC kit and Vector VIP kits; Vector Labs).

Primary goat endometrial epithelial cells (EECs) and stromal cells (ESCs) were isolated from the endometrium of 8-month-old goats (n = 6) on Day 7 of estrous cycle as previously described [11 (link)]. The primary endometrial cells were cultured and maintained in DMEM/F-12 (Gibco, Invitrogen Corporation, Grand Island, NY) supplemented with 10% charcoal stripped fetal calf serum (FCS; Biowest, France), antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Gibco) at 37°C in a 5% CO₂ –95% air atmosphere. The primary endometrial cells cultured on the cover slides were immediately fixed in 95% ethanol (v/v) and immunostained for cytokeratin and vimentin using an avidin-biotin complex (ABC) immunoperoxidase kit (Vectastatin Elite ABC kit; Vector Labs, Burlingame, CA) [11 (link)]. The protein expressions of 17β-estradiol receptor (ER) and progesterone receptor (PR) were analyzed separately in the EECs and ESCs using western blotting as previously described [12 (link)].

MUC1 expression was assessed by IHC. The anti-MUC1 antibody 4H5 (with specificity against abnormal hypoglycosylated MUC1-N terminal subunit) was used for the purposes of IHC, as previously described [42 ]. The IHC staining procedures were performed manually at room temperature using avidin-biotin-peroxidase complex methods (Vectastatin Elite ABC kit; Vector Lab, Burlingame, CA). After sample slides were washed with phosphate-buffered saline for 5 minutes, slides were blocked with normal serum with 3% bovine serum albumin for 10 minutes followed by incubation with the primary antibody, rinsed, and incubated with a biotinylated secondary antibody and washed again. Slides were incubated with the avidin-biotin complex for 1 hour and washed again. Chromogen was developed with 3,3-diaminobenzidine (DAB) (DAB substrate kit for peroxidase; Vector Lab). Automated quantitative analysis was used as a quantitative imaging analysis tool for marker expression [43 (link), 44 (link)]. Levels of MUC1 expression in areas of dysplasia, metaplasia, AIS, and carcinoma present within the same tissue sample were characterized independently. MUC1 expression levels were quantitated numerically with a score of 0, 1, 2, and 3, corresponding to negative, weak, moderate, and strong staining intensity, respectively.

To examine the expression pattern of ST2-positive cells and FoxP3-positive Tregs in the adenoma/CRC microenvironment, IHC staining was performed on 4-μm paraffin-embedded sections from controls, adenomas and CRC using the Vectastatin Elite ABC Kit (Vector Laboratories Inc, Burlingame, CA, USA) according to the manufacturer’s instructions and our previously published methods^{4 (link),5 (link)}. For epitope retrieval, deparaffinized and rehydrated slides were placed in a plastic jar with a boiling solution of 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) for 20 minutes. The following primary antibodies were used: rabbit anti-ST2 polyclonal antibody (working dilution 1:100; Thermo Fisher Scientific, USA) and mouse anti-FoxP3 monoclonal antibody (working dilution 1:100, Abcam, UK). Tissue sections were incubated with the primary antibodies at 4 °C overnight. Then, the chromogen 3-amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA, USA) was used to detect the target antigens, and the tissues were counterstained with Mayer’s hematoxylin. Previous known tumor sections positive for ST2 and FoxP3 were used as positive controls to confirm the immunoreactivity of ST2 and FoxP3 in each series of IHCs. To exclude background staining by nonspecific antibody binding, negative controls were included using isotype-matched antibodies in each IHC test.