Cy3 conjugated goat anti rabbit igg

Following transfection, HCT-116 and LoVo cells (3 × 10⁴) that adhered to the coverslips were fixed with 4% paraformaldehyde for half an hour at room temperature, then infiltrated with Triton X-100 (0.5%) for 15min, and subsequently blocked with 5% BSA solution. The cells were first stained with E-cadherin (cat. no. 3195; dilution 1:200; Cell Signaling Technology, Inc.) rabbit antibody followed by Cy3-conjugated goat anti-rabbit IgG (cat. no. P0183; dilution 1:1,000; Beyotime Biotechnology, Inc.), or first stained with the Vimentin (cat. no. 5741; dilution 1:200; Cell Signaling Technology, Inc.) rabbit antibody followed by FITC-conjugated goat anti-rabbit IgG (cat. no. P0186; dilution 1:1,000; Beyotime Biotechnology, Inc.). Primary antibodies were incubated in room temperature for 60 min. The second antibody was incubated in dark for 60 min. After incubating with first and secondary antibodies at room temperature, DAPI was applied for nuclear staining with 30 min. Finally, cells were tested with a DMI3000B inverted microscope (Leica Microsystems GmbH). All experiments were repeated three times.

A standard immunohistochemistry procedure was performed as described in our previous studies [25 (link), 26 (link)]. Briefly, after tissues were sectioned (10-μm thickness) on a cryostat (CM 1950; Leica), TG and TNC sections were blocked with 5% normal goat serum in phosphate buffer saline (PBS) plus 0.15% Triton X-100 for 1 h and then incubated overnight in a primary antibody against NeuN (neuronal marker, rabbit, 1: 500, Abcam) or calcitonin gene-related peptide (CGRP) (mouse, 1: 500, Abcam). The sections were washed with PBS and incubated in Cy3-conjugated goat anti-rabbit IgG (1: 500, Cell Signaling Technology) or Dylight488 goat anti-mouse IgG (1: 500, Multi Sciences) for 2 h at room temperature. The slices were viewed under an upright fluorescence microscope (Eclipse Ts, Nikon) and images were taken using a CCD camera (Cool Snap HQ2). Negative controls incubated with secondary antibody only did not display any positive staining (not shown).