Bx40f microscope

About 30% cultured HCT116 cells in 24-well plates containing a cover slip (8D1007, Wuxi Nest Biotechnology Co., LTD., Jiangsu, China) on each well were treated with shBCCIP or shYY1 for 72 h. Cells were then washed, fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.3% TritonX-100 in PBS buffer for 5 min. After incubating cells with p53 (1:50), p21 (1:100), and YY1 (1:100) primary antibodies at 37 °C for an hour, cells were stained with FITC- or TRITC-conjugated secondary antibodies (rabbit/green: 1:300, ZF-0311; rabbit/red: 1:300, ZF-0316; mouse/green: 1:300, ZF-0312; mouse/red: 1:300, ZF-0313). Cell nuclei were stained by DAPI containing Vectashield (Vector Laboratories, Inc. H-1200). Fluorescence images were observed with an Olympus BX40F microscope (Olympus Corporation, Tokyo, Japan).

Statistical analysis was performed using GraphPad 8.0.1. Flow cytometry data were expressed as percentages and median fluorescence intensity (MFI). A nonparametric test (Kruskal–Wallis test) was used for median comparisons and completed with Dunn’s multiple comparisons tests. In experiments involving histology or IHC, the figures shown are representative of the tissue sections. The Allred scoring system was used for IHC staining quantification [22 (link)]. Proportion scores were: 0, none; 1, less than one percent; 2, one percent to one tenth; 3, one tenth to one third; 4. one third to two thirds; 5, Two thirds or more. The intensity scores were: 1 for weak, 2 for medium, and 3 for strong. We evaluated 10 high-power fields (× 400 magnification) in each sample. Added the average of the intensity score and the proportional score as the Allred total score (range = 0–8). The image was captured with an Olympus BX40F microscope (Olympus). All statistical assessments were two-sided, and statistical significance was defined as P < 0.05.

Cells were cultured and grown to ~30% confluence in 24-well plates containing a coverslip (8D1007, Nest) on each well. Cells were then transfected with INO80 siRNA or INO80 shRNA and hArp8 siRNA using RNAiMAX (Invitrogen). 48 hours later, cells were washed by PBS buffer, fixed with 4% Paraformaldehyde (PFA) for 15 minutes at room temperature, and permeabilized with 0.5% TritonX-100 in PBS buffer for 5 minutes, followed by blocking with 1% bovine serum albumin in PBS for an hour at 37°C. Then, cells were washed for 5 minutes in PBS-T three times, and incubated with p21, α-tubulin or pericentrin primary antibodies (1:200–500) at room temperature, then stained with FITC-conjugated secondary antibodies (rabbit/green: 1:300, sc-2012; mouse/green: 1:300, sc-2010; rabbit/red: 1:500, A10040). Cell nuclei were stained by Vectashield with DAPI (Vecter Laboraries,Inc. Cat.No-H-1200). Fluorescence images were observed with Olympus BX40F Microscope (Olympus Corporation).

NSL3 wild type (WT) or NSL3-KO 293T cells were cultured to approximately 30% confluence in 24-well plates containing cover slips (8D1007, Nest) in each well. Then, 48 h later, the cells were immunostained according to a method previously described [58 (link)]. H4K16ac (1:400 dilution) primary antibody and FITC-conjugated secondary antibody (rabbit/green, 1:300, ZF-0311) were used. Cell nuclei were stained using Vectashield with DAPI (H-1200). Fluorescent images were observed under an Olympus BX40F Microscope (Olympus Corporation, Tokyo, Japan).

Nineteen coprolites from the midden area were available for analysis. Sample preparation and analysis was undertaken in the Ancient Parasites Laboratory at the University of Cambridge using our routine methodology (Anastasiou and Mitchell, 2013 (link)). A 1 g subsample of each coprolite was disaggregated (made into a liquid suspension) by adding 5 mL 0.5% trisodium phosphate to the subsample. Material within the size range of interest was isolated from the disaggregate using stacked microsieves with mesh measuring 300, 160 and 20 μm. The material trapped on the 20 μm sieve was washed free of the mesh. This would contain any helminth eggs present, as the typical size range of eggs from helminths in northern Europe is 20–150 μm (Garcia, 2016 : 1233). The suspension was centrifuged at 4000 rpm (3100 g) for 5 min, and the supernatant then removed. The remaining pellet was mixed with glycerol and the entire subsample was viewed on a digital light microscope (Olympus BX40F microscope with GXCAM-9 digital camera) at 400× magnification to visualize any preserved parasite eggs.

HCT116 cells, grown to ~30% confluence in 24-well plates containing a coverslip (8D1007, Nest) on each well, were transfected with pcDNA3.1 and Flag-MOF plasmids. Additionally, 48 h after transfection, cells were immunostained with YY1 (1:400 dilution) and MOF (1:200) primary antibodies, and FITC-conjugated secondary antibodies (1:300, Santa Cruz sc-2012) were used. Cell nuclei were stained with Vectashield with DAPI (H-1200) (Vector Laboratories, Inc., Burlingame, CA, USA). Fluorescent images were observed with an Olympus BX40F Microscope (Olympus Corporation, Miyazaki, Japan) with a silicon immersion 40× objective.