Las sp8 confocal microscope

Fluorescent microscopy was performed on a Leica LAS SP8 confocal microscope; confocal images were obtained using the Leica AF Lite system. Confocal images from the basal layer of the posterior midguts (sub-region R5a, https://flygut.epfl.ch/overview) where ISCs can be clearly visualized were taken under 40 × objective. Single layer image is shown. The quantification of p-H3⁺ cell per guts was undertaken by counting the p-H3⁺ cell numbers over the whole gut of indicated genotypes. The p-H3⁺ cell number quantification data were statistically present as average with the standard error of mean (SEM) and p-values of significance is calculated by Student’s T-test (tails = 2, Two-sample unequal variance): * is p<0.05, ** is p<0.01, *** is p<0.001, ns is no significance with p>0.05.
To quantify the expression of indicated protein in Figure 5 and Figure 6, the intensity of the indicated proteins and GFP signal in the view region was analyzed using the Leica AF Lite system. For each group, 50 (GFP⁻) of which were quantified as wild type and 50 (GFP⁺) were quantified as lola^5D2 or wts^x1. The quantification data were statistically present as average with the standard error of mean (SEM) and p-values of significance is calculated by Student’s T-test (tails = 2, Two-sample unequal variance): * is p<0.05, ** is p<0.01, *** is p<0.001, ns is no significance with p>0.05.

HEK293 cells grown on cover-slip were transfected with required plasmids for 48 h and then treated with indicated chemicals followed by fixation with 4% paraformaldehyde (PFA) in PBS for 10 min. Cells were permeabilized and blocked with PBS/0.2% Triton X-100/1% BSA for 45 min. Cells were then incubated with indicated primary antibodies for 2 h at RT. After washing with PBS/1% BSA for three times, cells were incubated with Cy3-labeled goat anti-mouse or rabbit IgG secondary antibodies in the dark for 1 h, washed with PBS/1% BSA, and mounted on slides. Images were acquired using LAS SP8 confocal microscope (Leica, Germany) with a 63 ×/1.40 NA oil objective (Leica).
For the analysis of receptor internalization, the acquired images were subject to the measurement of fluorescence intensity at regions of plasma membrane and cytosol using ImageJ (http://rsb.info.nih.gov/ij/). The index of receptor internalization was then calculated according to the published equation [27 (link)]
To quantify the degree of colocalization between fluorophores, the images were background subtracted and subjected to the analysis of Mander’s colocalization coefficients using ImageJ (http://rsb.info.nih.gov/ij/).

U-2 OS cells cultured on glass cover slips were transiently transfected with designated constructs using Fugene HD Transfection Reagent (Roche, Basel, Switzerland). Twenty-four hours after transfection, cells were fixed 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized and blocked with PBS containing 0.2% Triton X-100 and 1% bovine serum albumin. Cells were next incubated with primary antibodies against calnexin, GM130 or EEA1. After washing in PBS containing 1% bovine serum albumin, cells were incubated with Cy3- or Cy5-labeled goat anti-mouse or rabbit IgG secondary antibodies in the dark. Cells were washed again and finally mounted on slides. Images were acquired on a LAS SP8 confocal microscope (Leica, Wetzlar, Germany) with a 63×/1.40 NA oil objective (Leica). Floating coronal ice sections of the mouse brains (30 μm thick) were incubated with antibodies against Aβ plaques (6E10) and astrocytes (GFAP) at 4 °C for 12 h. Images were captured using an AxioVert inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and quantification was performed using Image-Pro Plus 5.1 software (Media Cybernetic, Rockville, MD, USA).