Tissue staining kit

CRC and paired normal colorectal tissues were fixed using formalin, embedded in paraffin, cut into 5 μm sections, and stained using IHC [17 (link)]. The sections were incubated using the anti-TYRO3 (Abcam, UK) and anti-ENO1 (Boster, China) antibodies at 1:100 dilution for 2 hours at room temperature. TYRO3 expression in tissues was visualized with a tissue staining kit (Zhongshan Biotechnology, China). Two authors with expertise in pathology assessed the staining scores. IHC score was determined by the intensity multiple (0, negative; 1, weak; 2, moderate; 3, strong) and extent (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, >75%) score. We considered a final score of 0 as −; 1–4 as +; 5–8 as ++; 9–12 as +++. In this study, ++ or +++ was considered a positive expression, and – or + a negative [18 (link)].

Tissue specimens were fixed with 10% formalin, embedded in paraffin, and cut into 5μm-thick sections. After cleaned in xylene and rehydrated through an ethanol gradient, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidases, and then boiled in 10mM citrate buffer (pH 6) for antigen retrieval. The processed sections were then blocked with 10% goat serum for 30 min, and incubated overnight with 1:200 diluted polyclonal anti-human AKR1B10 (BOSTER, Wuhan, China) or anti-human FGF1 (BOSTER, Wuhan, China) at 4°C. Color was developed using a tissue staining kit (Zhongshan Biotechnology, Beijing, China). The AKR1B10 or FGF1 staining scores were evaluated in five random fields per slide by two pathologists YuHong Wang (The First Affiliated Hospital of Soochow University) and Zheng Zhi (The Soochow University) in a blinded manner as previously described [24 (link)]. The percentage of positively stained cells was scored as follows: 0 - 0-5%; 1 - 6-25%; 2 - 26-50%; 3 - 51-75%; 4 - >75%. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The final score was the average of the percentage score multiplied by intensity score, and graded as follows: – (0), + (1-4), ++ (5-8) and +++ (9-12). Samples with final scores ++ or +++ were graded as positive, and – or + as negative.

Surgical specimens were fixed in 10% formalin and embedded in paraffin. Briefly, the paraffin-embedded tissues were serially cut into 5 μm sections and incubated with the polyclonal antibody recognizing human DAB2IP at 1 : 200 dilutions 4°C overnight. The proteins were visualized using a tissue staining kit (Zhongshan Biotechnology, Beijing, China), and staining scores were examined by two clinical pathologists. Five random regions were analyzed and the presence of brown-colored granules on the cytoplasm was taken as a positive signal. The staining was divided by color intensity into not colored, light yellow, brown, and tan and is recorded as 0, 1, 2, and 3, respectively. Positive cell rate of <25% was a score of 1, positive cell rate of 25–50% was a score of 2, positive cell rate of 51–75% was a score of 3, positive cell rate of >75% was a score of 4. An average intensity score of 4 or above was considered as high expression and a score below 4 as none or low expression.

Fix the surgical specimens with formalin and embed them with paraffin. Then the tissues were cut into 5 μm. After dewaxing and rehydrating, sections were incubated with the polyclonal antibodies of Gli1 or HER2 (Cell Signaling Technology, USA; 1:200 dilution) at the room temperature for 2 or 3 hours. The process was performed with the tissue staining kit (Zhongshan Biotechnology, Beijing, China) following the manufacturer’s protocol. Two researchers got the outcome of immunostaining respectively. Five 200 × random regions were analyzed and classified into five levels according to the percentage of positively staining cells per section: absent, 0~5%; 1, 6~25%; 2, 26~50%; 3, 51~75%; 4, >75%. We deemed the staining intensity as follows: 0 (negative); 1 (weak); 2 (moderate); 3 (strong). The percentage and intensity scores were then multiplied to obtain a total score (staining score = percentage score × intensity score). For the staining score, 0 was deemed as (−), 1~4 as (+), 5~8 as (++), 9~12 as (+++). Eventually, we obtained a final average score, and consider (−) or (+) as negative, (++) or (+++) as positive^{41 (link)}.