Slowfadetm gold anti fade reagent

After ISS, the cover slips were removed from the glass slides, the tissue sections were washed in PBS, and TB Auramine–Rhodamine T (AR) staining was performed using the TB Fluorescent staining kit T (BD). Briefly, the tissue sections were incubated at 37°C for 15 min in pre-warmed TB AR (BD), washed with tap water, and decolorized for 4 min at room temperature with Decolorizer. After being washed with tap water, the tissue sections were stained with DAPI for 15 min in room temperature, washed with tap water, and air dried, and cover slips were mounted with SlowFadeTM Gold anti-fade reagent (Invitrogen). Imaging was performed in an Axio Imager Z2 epifluorescence microscope (Zeiss) with 20× objective by acquiring Z-stacks of overlapping tiles that together cover the tissue section (10% overlap).

Adult fly abdomens were dissected from flies and fixed with 4% formaldehyde in a fixative buffer (100 mM 1,4-piperazinediethanesulfonic acid (PIPES), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, and 2 mM MgSO₄, pH 6.9) for 30 min at 25 °C. For Nile Red staining, flies were washed in phosphate-buffered saline with Tween 20 (PBST) and incubated for 30 min in Nile Red (Sigma-Aldrich, Cat#: M3013, St. Louis, MO, USA) staining solution (1 mg/mL in dimethyl sulfoxide, 1:2000 in PBST). The samples were washed in PBST and mounted in SlowFade^TM Gold antifade reagent (Invitrogen, Cat#: S36936, Carlsbad, CA, USA).
For analysis of nephrocyte morphology, dissected abdomens were fixed with a fixative buffer for 30 min and washed in PBST. The samples were mounted in an antifade reagent. All images were collected using a Leica fluorescence microscope (MZ10F) (Leica Microsystems, Wetzlar, Hessen, Germany) and a Carl Zeiss confocal microscope (LSM710) (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany).

Liver samples were fixed in 4% paraformaldehyde, processed for paraffin embedding, and cut at 7 μm thickness. The slides were de-waxed in xylene, re-hydrated in graded ethanol and then subjected to antigen retrieval in 1X citrate buffer pH 6 (GBI Labs, Catalog# B05C-100B) for 15 min at 110 °C and 5 psi. Slides were cooled, blocked in 0.25% Casein (Sigma Aldrich, Catalog# C7078) solution (in PBS) for 10 min at room temperature and incubated with a 1:400 dilution of an anti-CD3 rabbit antibody clone SP7 (Epredia Lab Vision #RM9107S) for 1 hr at room temperature. Slides were washed in TBS-T for 5 min and incubated with 1:250 dilution of a goat anti-rabbit IgG (H + L) antibody (Invitrogen #A21244) for 1 h at room temperature. Slides were washed in TBS-T for 5 min and then stained with DAPI (Invitrogen #D1306) diluted to 1:10,000 in TBS-T for 5 min at room temperature. Slides were washed, mounted with SlowFade^TM Gold anti-fade reagent (Invitrogen, Catalog# S36937) and cover-slipped. Images were acquired by scanning the entire tissue at Å~40 magnification using an Olympus VS120 Slide Scanner, pseudocolored, and analyzed using the CellSens™ Dimension Desktop v1.18 software (Olympus).