Sp 9000

Immunohistochemical staining was performed using Biotin-Streptavidin horseradish peroxidase (HRP) Detection Systems (SP-9000, ZSGB-BIO, Beijing, China). The tissue sections were incubated with 3% H₂O₂ deionized water for 10 min and blocked with goat serum (SP-9000, Reagent A) at room temperature. Then, the tissue sections were incubated with rabbit polyclonal anti-KLF4 (1:2,000 dilution, Cat. No. ab215036; San Francisco, Abcam), rabbit polyclonal anti-E-cadherin (1:400 dilution, Cat. No. 3195T; Danvers, CST), rabbit polyclonal anti-vimentin (1:3,000 dilution, Cat. No. 10366-1-AP; Rosemont, Proteintech) primary antibodies overnight at 4°C.
Immunofluorescence staining was performed to detect KLF4, E-cadherin, and vimentin expression. The tissue sections were blocked with 5% bovine serum albumin (BSA) and incubated with rabbit monoclonal anti-KLF4 (1:500 dilution, Cat. No. ab214666; San Francisco, Abcam), rabbit polyclonal anti-E-cadherin (1:200 dilution, Cat. No. 3195T; Danvers, CST), and rabbit monoclonal anti-vimentin (1:400 dilution, Cat. No. ab92547; San Francisco, Abcam) primary antibody overnight at 4°C separately. The tissue sections were incubated with GoatAnti-Rabbit IgG H&L(HRP) (1:300 dilution, Cat. No. ab6721; San Francisco, Abcam) second antibody at room temperature for 1 h.

After deparaffinization, the slices were rehydrated; washed with PBS; incubated with 3% hydrogen peroxide at room temperature for 15 minutes; washed with PBS; incubated with hyaluronidase IV at 37 °C for 30 minutes; washed with PBS; blocked with 1:10 normal blocking serum at room temperature for 45 minutes; and incubated with a 1:100 dilution of anti-COL-I (ab84956, Abcam), a 1:100 dilution of anti-Osterix (ab22552, Abcam) or a 1:100 dilution of anti-COL-X (ab58632, Abcam) at 4 °C overnight. The slides were washed and incubated with a secondary antibody (sp9000, ZSGB-BIO) at 37 °C for 20 minutes, washed with PBS incubated with streptavidin peroxidase at 37 °C for 20 minutes. Then, the slides were washed with PBS and exposed for 5 minutes to the peroxidase DAB substrate (ZSGB-BIO, China). After staining with hematoxylin, the slides were examined with an Olympus microscope mounted with an Olympus video camera. We used Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) to analyze the results. We first circled the area of interest and then measured the cumulative optical density (OD) of what we stained. Afterward, we determined the average OD with the cumulative OD divided by the size of the area of interest.

Immunohistochemistry was performed to test target proteins expression in samples from clinical patients and tumour xenographs by standard protocols. Briefly, paraffin sections (4 μm) were deparaffinized and rehydrated, and heat-induced antigen retrieval was conducted in sodium citrate buffer (10 mM, pH 6.0). Endogenous peroxidases were blocked by incubation in 0.3% H₂O₂. The sections were then incubated with primary antibodies against FMNL3 (Cat. #HPA023201; Sigma, Germany), E-cadherin (Cat. #3195 S; Cell Signaling, MA, USA), Vimentin (Cat. #5741 S; Cell Signaling) and MMP-9 (Cat. #sc-21733; Santa Cruz) at 4 °C overnight. Non-immune IgG was used as a negative control. Antigenic sites were visualized using a SP9000 and DAB kits (ZSGB-BIO, Beijing, China). The immunoreactive scores (IRS) of FMNL3, E-cadherin, Vimentin and MMP-9 were calculated as follows: 0, negative; 1, weak; 2, moderate; or 3, strong. The percentage of positive cells was scored as follows: 1, 0–9% positive cells; 2, 10–50% positive cells; and 3, >50% positive cells. The two scores were multiplied together and samples with a total IRS of 0, 1–3, 4–6 and 7–9 were considered as (−), (+), (++) and (+++), respectively.