Rm2126

The treated fourth instar larvae were fixed in 4% paraformaldehyde for 24 h. The fixed samples were prepared according to the standard method [25 (link)]. The prepared samples were cut using the rotary microtome (Leica RM2126, Wetzlar, Germany), stained with hematoxylin-eosin and observed by a light microscope (Olympus, BX51, Tokyo, Japan). The ventral muscle tissues, Malpighian tubules and midgut digestive cells of treated larvae were observed and compared to control.

Analysis of CD8⁺ T cells infiltration in ICT-treated tumor tissues by immunofluorescence. Firstly, the tumor tissues were formaldehyde-fixed and paraffin-embedded following standard procedures. Then, paraffin tissue sections of 4 μm thickness were prepared using a paraffin slicing machine (LEICA RM2126) and the sections were roasted overnight at 37° C, dewaxed and rehydrated. They were then sealed with goat serum for 30 min, washed 3-4 times in PBS, and incubated overnight at 4° C with CD8 Polyclonal Antibody (PA5-88265, Thermo Scientific) and FOXP3 Monoclonal Antibody (11-5773-82, Thermo Scientific). After washing 3-4 times with PBS, the Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-21245, Thermo Scientific) was added dropwise, incubated at 37° C for 1 h, stained with DAPI for 5-10 minutes, washed 3-4 times with PBS, and air-dried. Observed under a confocal microscopy (Nikon A1R).

The paraffin block containing tissues was trimmed into a small cuboid and cut into 6- to 12-μm slices by a microtome (Leica RM2126).