Formaldehyde

Germarium preparation for whole-mount immunofluorescence was performed as previously described (Page and Hawley 2001 (link)). Two to 4-day-old females were collected and yeasted overnight at 24 °C. Ovaries were dissected in under 10 min in PBS with 0.1% Tween (PBST). Ovaries were fixed in 200 μL of PBS containing 2% formaldehyde (Ted Pella, Redding, CA) and 0.5% Nonidet P-40 plus 600-μL heptane at room temperature for 20 min. Then the ovaries were washed three times in PBST for 10 min. Ovarioles were teased apart with forceps and the late stage egg chambers were removed before being blocked for 1 h in PBST with 1% bovine serum albumin (BSA) (EMD Chemicals, San Diego, CA). Ovarioles were incubated overnight with primary antibody diluted in PBST at 4° while nutating. The ovarioles were then washed with PBST three times for 20 min. Ovarioles were incubated for 2 h in secondary antibody in PBST and 4′6-diamididino-2-phenylindole (DAPI) (final concentration of 1 μg/ml) was added for the final 10 min. The ovarioles were washed in PBST three times for 15 min and then mounted in ProLong Gold (Life Technologies, Grand Island, NY). The primary antibody used was affinity-purified rabbit anti-Corolla (1:2000). The secondary antibody, Alexa Fluor 488 goat anti-rabbit (Thermo Fisher, A11008), was used at 1:500.

Five litters of each group with ~ 20,000 PGCs (Zombi Red⁻/SSEA-1⁺/CD61⁺) were designated to proceed for Hi-C sample preparation using Arima Hi-C Kit following the manufacturer’s instructions for low-input Hi-C sequencing. This low-input protocol supported quantitative determination of topologically associating domains (TADs) from 10,000 human cells (Arima Genomics Application Note: “Unlock Low-Input 3D Genome Analysis with the Arima-HiC Kit”, Arima Genomics), which was confirmed by our current study (Extended data Fig. 2a-2d). Briefly, cells were fixed with formaldehyde (Ted Pella) to crosslink chromatin contacts. Genomic DNA was isolated from the fixed cells and digested using a restriction enzyme cocktail. The digested 5’-overhanging ends were filled with biotinylated nucleotides, and spatially proximal digested ends were ligated. Proximally ligated DNA fragments, which capture chromatin contacts were purified, fragmented by sonication, and enriched using streptavidin-conjugated beads. Illumina sequencing libraries were synthesized from the solid phase-captured DNA fragments using the Swift Biosciences^® Accel-NGS^® 2S Plus DNA Library Kit (Swift). Libraries were sequenced using an Illumina NovoSeq 6000 deep sequencer to obtain 150 + 150 nt paired-end FASTQ reads.

Sterilized gold micro-patterned glass coverslips were functionalized with poly-D-lysine in hydrobromide solution (70 μg/ml; Sigma, P6407) for 1 hour and rinsed off with ddH2O. Mouse hippocampal neurons were prepared as previously described42 (link). In brief, primary neurons prepared form E18 mice embryos were plated onto functionalized gold micro-patterned coverslips using NBM (Neurobasal Medium, Gibco, 21103-049), supplemented with B27 (Gibco, 17504-044), Pen/Strep (Biochrom AG, 12212) and 20 mM HEPES (Gibco, 15630). For immunostaining, cells were fixed 8 days after plating (DIV 8) in 1× PBS (Gibco, 10010-023) containing 4% Formaldehyde (Ted Pella, 18505) and 120 mM sucrose for 20 minutes at room temperature and quenched for 20 minutes with 100 mM NH₄Cl (Carl Roth, K298.2). Then blocking and permeabilization was performed with 0.1% Triton-X100 (Sigma, T9284) in PBS containing 2.5% bovine serum albumin (Sigma; A9085.25G) for 15 minutes at room temperature. The cells were incubated for 1 hour at room temperature with mouse monoclonal anti-MAP2 antibody (1:250; Synaptic Systems, 188 011) and Hoechst 34580 (2 μg/ml; Life Technologies, H21486). Primary antibodies were detected using goat anti-mouse 488 (1:1000: Invitrogen, O-6380).

For antibody staining, imaginal disks and brains were collected from third instar larvae and fixed for 23 min [0.1 M PIPES (pH 6.9), 1 mM EGTA (pH 6.9), 1.0% Triton X-100, 2mM MgSO4, 4% formaldehyde (Ted Pella, Inc.)]. Fixed tissues were blocked for 2 hrs at 4°C with blocking buffer (1X PBS, 0.1% Tween-20 and 5% normal goat serum). Tissues were incubated over night at 4°C with primary antibody diluted in blocking buffer (rabbit anti-En 1:500 dilution, Santa Cruz Biotechnology, Inc.; mouse anti-ßgal 1:500 dilution, Invitrogen). After removal of primary antibody, tissues were washed (1X PBS, 0.1% Tween-20) four times- 15 min each. Washed tissues were incubated in fluorescent tagged secondary antibody diluted in blocking buffer for 2 hrs at 4°C in dark, washed 3 times and mounted with DAPI-Vectashield (Vector Laboratories). All wing imaginal discs were quantified by ImageJ.

Cells grown on coverslips were fixed with cold methanol for 8 min at −20°C or 4% formaldehyde (Ted Pella, Inc) for 15 min at room temperature. Cells fixed with formaldehyde were permeabilised with 0.1% Triton in PBS and then incubated with blocking reagent (Roche) or 2–5% BSA for 30 min at room temperature. Primary antibodies were diluted in blocking reagent or 2% BSA and incubated overnight at 4°C or at 37°C for 1 hr. Secondary antibodies conjugated with Alexa Fluor 594, 488, or 647 were diluted in blocking reagent and incubated for 1 hr at room temperature.
Confocal images were taken with a TCS SP5 (63×, 1.4–0.6 NA, oil, HCX PL APO), TCS SP8 (63×, 1.4 NA, oil, HC PL APO CS2), all from Leica Microsystems, using Leica acquisition software. Lasers and spectral detection bands were chosen for the optimal imaging of Alexa Fluor 488, 594, and 647 signals. Two-channel colocalization analysis was performed using ImageJ (National Institutes of Health).