Dm4000b microscope

As described previously (Manitt et al., 2013 (link); Reynolds et al., 2018 (link)), the density of TH-positive (TH+) fibers and varicosities in the inner layers of the PrL and IL regions of the pregenual mPFC was evaluated using a stereological fractionator sampling design (West et al., 1991 (link)), with the optical fractionator probe of the Stereoinvestigator software (MicroBrightField). In the PFC, the TH antibody labels predominantly DA axons and rarely labels norepinephrine axons (Manitt et al., 2011 (link), 2013 (link)). Regions of interest were delineated according to the mouse brain atlas (Paxinos and Franklin, 2019 ), and contours of the TH+ projection within these regions were traced at 5× magnification with a Leica DM4000B microscope. Stereoinvestigator calculates, for each brain region, a volume (in cubic micrometers) measurement from the contour area, section thickness, and section periodicity (MicroBrightField). Sections spanning plates 14–18 of the mouse brain atlas were studied. Stereoinvestigator calculates the total number of TH+ varicosities based on the experimenter’s random sampling of a known fraction of the region. Counting frame and grid size were chosen to consistently sample 33 sites per region.

All specimens are deposited in the Insect Collection, Seoul National University (SNU), South Korea. Digital images of dorsal habitus are taken with a Canon EOS 70D, with a Canon MP–E 65–mm F2.8 1–5x macro lens. Genital structures are dissected and observed under a Leica DM 4000B microscope, and images are taken using a digital camera attached to the microscope (Lumenera Infinity 3). All measurements (mean and range) are provided in millimeters.
Terminology used to describe the male and female genitalia follows Yasunaga (1998) (link) and Yasunaga and Schwartz (2007) , and is indicated with the following abbreviations: IRL: interramal lobe; LL: lateral lobe; DLP: dorsal labiate plate; DOS: dorsal sac; FP: fin-like process (of theca); LS: left lateral sclerite; MS: median sclerite; PB: Phallobase; PML: primary lobe; RM: ramus; RS: right lateral sclerite; SD: seminal duct; SGP: secondary gonopore; SP: spiculum; SPGC: sclerotized perimeter of genital chamber; SR: sclerotized ring; TH: phallotheca.

In vitro IEC-6 cells were post-fixed in 4% paraformaldehyde for 20 min and incubated in 0.3% Triton X-100 for 20 min. Intestinal tissue sections or cells were washed 3 times in PBS, blocked with 5% BSA solution for 1 h, and incubated with a primary antibody overnight at 4 °C. The primary antibodies were as follows: anti-rabbit β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit GSK-3β (Santa Cruz Biotechnology, Santa Cruz, CA). An Alexa Fluor 594 secondary antibody (Invitrogen Life Technologies, Carlsbad, CA, USA) was added for 1 h at 37 °C. Then, 4,6-diamidino-2- phenylindole (DAPI, Beyotime, Shanghai, China) was added as a nuclear counterstain. A Leica DM 4000B microscope or a Leica TCS SP5 microscope was used to examine staining for conventional or confocal imaging, respectively.

Formalin-fixed and paraffin-embedded tissues were processed and sectioned according to routine protocols. Heat mediated antigen retrieval was used prior to all staining procedures. Tissues were incubated with vimentin antibody (1:200 dilution, clone D21H3, Cell Signaling Technology) or S100A9 antibody (1:100 dilution, provided by Dr. Philippe Tessier at Laval University) overnight at 4 °C. Antigen was detected using the EnVision+ Dual Link System (Dako) and counterstained with hematoxylin. Images were taken using a Leica DM4000B microscope and a Leica MC120 HD camera with a 40× objective.