Fluromount g

Once mice had completed all behavioral experiments, animals were perfused to verify GRIN lens placement and virus expression. Mice were deeply anesthetized and intracardially perfused with 4% paraformaldehyde in PBS. Brains were dissected and post-fixed with the same fixative. Coronal sections (40 μm) of the entire hippocampus were cut using a vibratome and sections were mounted directly on glass slides. Sections were split and half of all sections were stained for cresyl violet to localize GRIN lens placement, the other half of sections were stained for DAPI and mounted with Fluromount-G (Southern Biotechnology) to evaluate virus expression.
To evaluate the hm4di-mCherry fluorescence for each mouse, pyramidal cells were quantified across eight coronal slices representing the injection sites along the septo-temporal axis of the hippocampus, with three randomly selected images taken across three subregion (CA1, CA3, dentate gyrus) for a total of 72 images analyzed per animal. To evaluate the level of expression in each subregion, the number of transfected pyramidal cells was counted using imageJ (version 1.51j8) and virus expression was imaged with an AxioObserver.Z1 microscope (Carl Zeiss).

Embryo fixation and antibody staining were performed as previously described [7 (link)]. The following antisera were used at the indicated dilutions: rat dCREB antiserum at 1:10 000; rat antisera to DE-cadherin at 1 : 400 (Developmental Studies Hybridoma Bank, DSHB; Iowa City, IA); rabbit aPKC antiserum (Santa Cruz Biotechnology, Dallas, TX) at 1 : 500; mouse β-galactosidase (β-gal) antiserum (Promega, Madison, WI) at 1 : 500. Appropriate AlexaFluor 488-, 647- or rhodamine- (Molecular Probes, Eugene, OR) conjugated secondary antibodies were used at a dilution of 1 : 500 for salivary glands. Anti-mouse-Cy5 secondary antibody was used at 1 : 200 dilution for wing discs (Jackson, West Grove, PA). Stained embryos were mounted in Aqua Polymount (Polysciences, Inc., Warrington, PA) and thick (1 µm) fluorescence images were acquired on a Zeiss Axioplan microscope (Carl Zeiss) equipped with an LSM 710 for laser scanning confocal microscopy.
To collect wing discs, the larvae were dissected in 1× phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature, washed with 1× PBS for 10 min, followed by a 5 min wash with PBTx (0.1% Triton X-100). The wing discs were stained with 10 µg ml⁻¹ Hoechst33342 in PBTx for 2 min, washed three times in PBTx and mounted on glass slides in Fluromount G (SouthernBiotech).

Paraffin-embedded slides were dewaxed in Histoclear and rehydrated in ethanol. H₂O₂ treatment was omitted. To block nonspecific binding, slides were incubated in 15% goat serum for 30 minutes. Rabbit anti–E. coli primary antibody (1:1000; Abcam, Cambridge, UK) was added and slides were left overnight at 4°C. Sections were incubated for 2 hours with a fluorescent goat anti-rabbit secondary antibody (1:1000, Alexa Fluor 488; Invitrogen, Carlsbad, CA). The sections were treated with DAPI (5 mg/mL) in the dark for 2 minutes and were transferred to ice-cold TBS. The slides then were left to dry followed by application of a coverslip with Fluromount G (Southern Biotech, Birmingham, AL).

For IF, cells were grown on coverslips and fixed with 100% ice-cold methanol for 5 min at −20°C. Cells were washed and then blocked in 1% BSA in PBS (v/v) for 1 h, before successive incubation with primary antibodies (overnight at 4°C) and then secondary antibodies (1 h at room temperature). Coverslips were mounted onto glass slides using Fluromount-G (Southern Biotech) along with Hoechst 33342 (1:5000) for nuclear staining, or a droplet of ProLong Gold anti-fade reagent containing the DNA stain DAPI. Staining of endogenous Tiam1 was performed using a protocol described by Whalley et al. (2015) (link), which was shown to specifically detect centrosomal Tiam1.

To collect wing discs, the larvae were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature, washed with PBS for 10 minutes, followed by a 5 min wash with PBT (0.1% Tween-20). For antibody staining, wing discs were permeabilized in PBT (0.5% Tween-20) for 10’ and rinsed in PBT (0.1% Tween-20). The discs were blocked in 5% Normal Goal Serum in PBT (0.1% Tween-20) for at least 30 min and incubated overnight at 4°C in primary antibody in block (1:100, mouse monoclonal anti-Wg, Developmental Studies Hybridoma Bank Cat# 4D4; undiluted mouse monoclonal anti-Myc, Developmental Studies Hybridoma Bank Cat# P4C4-B10). The discs were rinsed thrice in PBT (0.1% Tween-20) and incubated in secondary antibody at 1:500 dilution in block for 2 h at room temperature. The wing discs were stained with 10μg/mL Hoechst33342 in PBT (0.1% Tween-20) for 2 minutes, washed 3 times in in PBT and mounted on glass slides in Fluromount G (SouthernBiotech). Wing discs were imaged on a Leica DMR compound fluorescence microscope using a Q-Imaging R6 CCD camera and Ocular software. The images were assembled, processed, and quantified using Image J software.