Bx3 cbh microscope

The paraffin blocks of tumorous and paracancerous renal tissues were cut into 4-μm slices. The slices were boiled in citrate buffer (0.01 M, pH 6.0) in a microwave oven using high power for 4 min, and cooled to RT. After doing this four times, the slices were washed clean with PBS for 3 × 5 min, blocked in 3% H₂O₂ for 20 min and in 10% nonimmune goat serum for 15 min at RT, and incubated with FAM129A antibody (1:200) at 4 °C overnight. The tissue slices were then warmed at 37 °C for 30 min, treated with biotin-streptavidin HRP detection kit (ZSGB-BIO, China), and imaged with 3,3′-diamino-benzidine (DAB) development kit (ZSGB-BIO, China) under an upright light BX3-CBH microscope (Olympus, Japan).
The degree of IHC immunoreactivity was judged by multiplying the staining immunoreaction intensity (Score I) and the DAB positive staining quantity (Score II) of tumor cells as previously reported^{33 (link)–35 (link)}. Score I was classified into four grades, 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Based on DAB positively stained cells, Score II was rated as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%). Immunoreactivity degrees with scores of 0–2, 3–5, 6–8 and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++) expression of FAM129A. IHC assays were separately scored by two experienced pathologists.

IHC assay was done to determine the expression changes of ANXA5 (Proteintech, China), RAC1 (Proteintech, China) and CRKI/II (GeneTex,USA) in HCC tissue array, and of CD34 and VEGF-3 (SantaCruz, USA) in primary tissues from Hca-P-ANXA5-shRNA1 and Hca-P-ANXA5-shControl-transplanted mice. Tissue sections were treated with biotin-streptavidin HRP detection systems (ZSGB-BIO, China) according to the manufacturer’s protocol. The images were visualized with 3,3′-diamino-benzidine (DAB) kit (ZSGB-BIO, China) under an BX3-CBH microscope (Olympus, Japan).
IHC immunoreaction intensity was rated into four grades, 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) as Score I. Moreover, based on the detected positively staining cells, DAB staining quantity of each sample was graded as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%) as Score II. The multiplication of Score I by Score II ranged 0 to 12 was used for IHC immunoreactivity degree. The scores of 0–2, 3–5, 6–8, and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++). IHC assay was scored by two independent experienced pathologists, separately.