Pannoramic midi automatic digital slide scanner

Rat brain tissues were harvested under anesthesia with perfusion of 4% paraformaldehyde (pH 7.4) as before [19 (link)]. All brain tissues were collected carefully without disruption of intact brain. Frozen coronal slices (8 μm thickness) of rat brain were prepared in the cryostat (CM3050S; Leica Microsystems, Bannockburn, IL, USA). TritonX100 and blocking serum of 0.2% (w/v) were successively added into brain sections with incubation of 15 min and 2 h, respectively. With incubation in the specific primary antibodies at 4 °C overnight (see Supplementary Table 1), corresponding secondary antibodies (Alexa 488/594-conjugated or Cy3-conjugated) for 2 h at room temperature without light, and nuclei staining with DAPI for 10 min, brain sections were stained with immunofluorescence. The immunofluorescence was examined under FLUOVIEW FV1000 confocal laser scanning microscope (Olympus, Japan) or Pannoramic MIDI automatic digital slide scanner (3D HISTECH, Hungary).
BrdU immunostaining was performed as described [19 (link), 24 (link)]. BrdU (Sigma-Aldrich, St. Louis, MO) was injected every other day until death (50 mg/kg, IP, dissolved in saline).

Slides ware deparaffinized and dehydrated by graded alcohol. Antigen retrieval was performed by boiling the slides in 1 mmol/L sodium citrate buffer pH 6.0 and then at sub‐boiling temperature for 20 minutes. Endogenous peroxidase activity was blocked by incubating the slides with 3% H₂O₂ for 10 minutes. Slides were washed in PBS, blocked with serum for 30 minutes, and incubated with a rabbit antibody for IQUB (1:400 dilution; Biorbyt) at 4°C overnight in a humidified container. After washing 3 times with PBS, the corresponding anti‐rabbit secondary antibody was incubated with a 1:300 dilution for 2 hours at 37°C. Staining was performed using 3, 3‐diaminobenzidine (DAB; Vector laboratories) for 2 minutes, and the colored reaction was terminated using distilled water. Slides ware counterstained with hematoxylin and Eosin Staining Kit (Beyotime Biotechnology Co.) to visualize cell nuclei, followed by bluing in running tap water. Finally, the slides were dehydrated, dipped in xylene and mounted. The Pannoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, Hungary) was used for image processing and quantifications. The protein expression levels of IQUB were measured by the intensity of immunohistochemical staining which could be quantified using an IHC analyzer in Image J software.16

Immunohistochemistry was used to detect the expression level of the protein in esophageal tissue. The tissue chip was dewaxed, and after dehydration, the endogenous peroxidase in the tissue was inactivated using 1 mmol/L sodium citrate buffer (pH 6.0). After blocking with goat serum for 30 minutes, the antibodies of the protein of interest were separately incubated overnight at 4°C. The next day, the biotin‐labeled secondary antibody was incubated with the tissue chip for 1 hour at room temperature, followed by incubation with streptavidin‐labeled horseradish peroxidase for 1 hour and finally with 3, 3‐diaminobenzidine (DAB), and the depth of the color represents the expression level of the target protein. The Pannoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, HUNGARY) was used for image capture. Quantification of target protein staining was performed using IHC profiler in ImageJ.13

The protein expression level of USP45 was detected by immunohistochemistry in COADREAD (n = 64), ESCA (n = 36), GBMLGG (n = 38), LIHC (n = 47), LUAD (n = 63), LUSC (n = 38), OSCC (n = 63), PAAD (n = 47), PRAD (n = 43), RCC (n = 78), TNBC (n = 42), non-TNBC (n = 38) and their paired paracancerous tissues. All of the tissue microarrays were purchased from were purchased from Outdo Biotech company (Shanghai, China). The procedure for immunohistochemistry refers to our previous study (Li et al., 2020 (link)). The results processing of immunohistochemistry was performed by Pannoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). The intensity of immunohistochemistry staining was then quantitatively scored. On the one hand, the proportion score was quantified as followed: 0 represents 0% of tumor cells exhibited positive staining, one represents 0%–1% positive cells, two represents 2%–10% positive cells, three represents 11%–30% positive cells, four represents 31%–70% positive cells, and five represents 71%–100% positive cells. On the other hand, the intensity score was quantified as followed: 0 represents negative; one represents weak; two represents moderate; and three represents strong. The total score is calculated as the mean of the proportion score and the intensity score.