Peroxidase conjugated secondary antibody

The tumor tissues obtained from the mice that received treatment with NS, Blank NP, Sal free 2, Sal NP 2, and Sal NP 8 were selected for further study. These tissues were ground into cell suspensions. Cells were washed twice with phosphate-buffered saline (PBS) solution and lysed with RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) and protease inhibitor (Thermo Fisher Scientific, USA). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Equivalent amounts of total protein (60 µg) were boiled and electrophoretically separated on a 10% polyacrylamide gel at 80 volts. The proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked for 60 min with a 5% milk solution prepared in PBS, incubated overnight at 4°C with primary antibodies (GAPDH, CD44, CD133, E-cadherin, VIM, ZEB1, and ZEB2) at a 1:500 dilution. The membranes were washed thrice for 5 min each time with Tween 20 (1:1000 dilution)-PBS and incubated for 45 min with the appropriate peroxidase-conjugated secondary antibody (Abcam, USA) (1:1000 dilution). Membranes were washed with Tween 20-PBS thrice for 10 min each time and visualized using the Odyssey two-color infrared laser imaging system (ECDOI, Greenville, USA). The signal generated by GAPDH was used as an internal control.

Cells transfected with CCT4 shRNA and control shRNA were harvested, and the total protein was extracted using radio-immunoprecipitation assay (RIPA) buffer supplemented with fresh protease and phosphatase inhibitors. The protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (50 μg) were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in 10 mmol/L Tris-hydrochloride (HCl) (pH 7.4, with 0.05% Tween-20) and incubated with primary antibody at 4°C for 12 hours. After washing with Tris-HCl buffer three times, the membranes were incubated with a corresponding peroxidase-conjugated secondary antibody (Abcam, Cambridge, UK). The membranes were developed using Super-Signal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). The densitometry of the protein bands was quantified by ImageJ software (https://imagej.nih.gov/ij/). This experiment was repeated at least three times. The information about the antibodies is listed in Table 1.

Cells transfected with siRNA were harvested and total proteins were extracted from tumor cell lines using RIPA buffer containing fresh protease and phosphatase inhibitors. The protein concentration was determined using the BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Briefly, equal amounts of proteins (50 µg) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA in 10 mM Tris-HCl (pH 7.4) containing 0.05% Tween-20 and incubated with a primary antibody at 4°C for 12 h. After washing with Tris-HCl buffer for three times, the membranes were incubated with a corresponding peroxidase-conjugated secondary antibody (Abcam). Immunoreactive bands were visualized using Super-Signal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). The densitometry of the protein bands was quantified by ImageJ software (1.8.0; National Institutes of Health). The experiment was repeated at least three times. The details of the primary antibodies used in the experiments are documented in Table I.

Immunohistochemistry (IHC) staining was routinely carried out using an immunohistochemistry kit (abcam, UK). The tissue sections were deparaffinized and rehydrated. The antigen was retrieved by sodium citrate buffer. Following endogenous peroxidase blocking and incubation in normal goat serum (Vector Lab, Burlingame, CA) for 20 mins, these sections were incubated with ERO1A (1:200, LSBio, USA), CEA (Use directly, zsbio, China), CA19-9 (Use directly, zsbio, China) overnight at 4°C and then with the peroxidase-conjugated secondary antibody (abcam, UK). Color development was performed with 3,3′-diaminobenzidine (DAB) and slides were counterstained with hematoxylin. Neutral resin sealing was performed. The results of immunohistochemical staining were observed under a microscope (Olympus Tokyo, Japan).