See Additional file 1 for a detailed step-by-step protocol and Additional files 2, 3, 4 for illustration of key steps in the procedure.
Preparation of heterogeneous short interfering RNA (hsiRNA)
Total RNA was prepared from adult B. malayi (TRS Labs, Athens, GA, USA) by established methodology http://www.filariasiscenter.org/molecular-resources/protocols and ~ 700 ng RNA used as template for production of cDNA using the ProtoScript M-MuLV First Strand cDNA Synthesis Kit according to the manufacturer's instructions (New England Biolabs. Ipswich, MA, USA).
DNA templates for in vitro transcription were generated by PCR using Crimson Taq DNA Polymerase (New England Biolabs). PCR primers contained T7 promoter sequence followed by two guanine bases at their 5' ends for transcription by T7 RNA polymerase and enhanced transcription yield. Primers were designed to yield a PCR product corresponding to ~500 bp of the transcript selected for gene silencing. Typically a region towards the 5' end of the open reading frame was selected and primers that would span an intron in genomic DNA (gDNA) were designed. This allows differentiation between amplification from 1st strand cDNA and any residual gDNA present in the cDNA preparation. However, we also demonstrated that RNA transcribed from gDNA that included introns was effective in gene silencing (see par-1 example). Primers having potential to amplify other B. malayi genes or to generate amplicons with considerable sequence similarity to non-target transcripts were avoided. PCR mixes (50 μl) generally contained ~100 ng template cDNA, 0.4 μM each primer, 0.2 mM dNTPs and 0.25 μl (1.25 U) polymerase in 1 × buffer. Between 1 and 5 μl of the PCR product was loaded on to a 1.5% agarose gel to assess the quality and size of the amplicon.
dsRNA corresponding to the gene to be targeted by RNAi was prepared using the HiScribe T7 In Vitro Transcription Kit according to the recommended protocol (New England Biolabs). Approximately 1 μg of PCR product was used in a 80 μl reaction without any purification prior to transcription. The reaction was incubated at 42°C for 2.5 hours then checked by loading 1 μL on a 1.5% agarose gel alongside dsRNA Ladder-A (New England Biolabs). When suitable RNA products were visualized, the remainder of the sample was purified by isopropanol precipitation and the RNA pellet resuspended in 50 μL RNase-free water.
The long dsRNA was processed to heterogeneous short interfering RNA (hsiRNA) using ShortCut RNase III as recommended (New England Biolabs). The entire preparation of dsRNA (50 μl) was used in a 100 μl reaction. The hsiRNA was purified by ethanol precipitation, resuspended in 60 μL RNase-free water, and an aliquot examined by gel electrophoresis (2% agarose) alongside siRNA Marker (New England Biolabs). Quantification of hsiRNA was achieved using a Nanodrop or standard spectrophotometer.
In vitro hsiRNAi by soaking
In a laminar flow hood, the appropriate amount of hsiRNA was placed in wells of a 12-well cell culture plate to which 1 ml Worm Culture Medium (WCM) was added. [WCM: RPMI-1640 cell culture medium with L-glutamine (Invitrogen, Carlsbad, CA, USA), 1% glucose, 100 × antibiotic-antimycotic solution (A-5955; Sigma-Aldrich, St Louis, MO, USA), 10% fetal bovine serum inactivated at 56°C for 30 minutes (Invitrogen)]. Two female worms were transferred to each well using a curved pick (see Additional file 2). The medium was replaced every 12 hours by pipetting hsiRNA into new wells, adding fresh WCM and then transferring the worms (see Additional file 3). Typically 1 μM hsiRNA was used but the concentration was increased up to 5 μM in instances where no effect was observed. Controls included worms cultured similarly but in the absence of any RNA and worms cultured in an equivalent concentration of Lit28i polylinker ShortCut siRNA mix (New England Biolabs).
Worms were incubated in a 37°C, 5% CO2 incubator. Exposure to the hsiRNA mixture was continued for between 2 and 5 days.
Embryo and Tissue Collection and Fixation
We optimized and simplified our previously published protocol . The worms from each well were transferred to a drop of 10 μl 1 × PBS on a microscope slide using the curved pick. For maximizing recovery of embryos, the worms were diced into small fragments using a razor blade (see Additional file 4). To obtain larger anatomical structures, such as the ovaries, fewer cuts were made under a dissection microscope, close to the posterior tip for instance, since the hydrostatic pressure expels organs out of the pseudocoelom and into the PBS. Once the ovaries, uteri, or intestine were expelled, they were cut into shorter fragments to facilitate subsequent tissue penetration by antibodies. The fragments and embryos were transferred to a microcentrifuge tube by pipetting 180 μL 1 × PBS, 1% NP-40 across the slide surface and into the tube (see Additional file 4). Next, 20 μl 32% paraformaldehyde and 2 volumes heptane (~400 μl) were added. The tube was vortexed for one minute, then left on a rotator for 20 minutes. The contents were pelleted in a microcentrifuge (2000 × g, 1 min), the supernatant removed and the pellet resuspended in 1 ml PBST-BSA (1 × PBS, 0.02% Triton X-100, 2% BSA (Fraction V; Fisher Scientific, Pittsburgh PA, USA)) by rotation for 5 minutes.
For embryo staining specifically, the tube was centrifuged again and the pellet transferred to a microscope slide using a Pasteur pipette. A coverslip was applied and pressed down gently using a paper towel to absorb excess PBST. Using forceps, the slide was held submerged in liquid nitrogen until the liquid stopped boiling. The slide was placed on the bench and the coverslip removed with a razor blade. Once the embryos had thawed they were washed into a microcentrifuge tube by pipetting PBST-BSA over the slide surface (see Additional file 4). This "freeze-crack" step removes a high proportion of the eggshells, thereby increasing the yield of high quality immunostaining.
These protocols were optimized for young embryos and tissue fragments, and permit immunostaining of all embryos up to about the 20 cell stage. However, the proportion of stained embryos decreases with increasing development stage, possibly because of changes in the eggshell composition, that increase resistance to the treatment.
Alternative treatment for fixation of older embryos
To increase the staining of older embryos (i.e. after morphogenesis begins), the following step was used instead of the "freeze-crack" procedure. After dicing the worms, the tissues/embryos were transferred from the slide to a microcentrifuge tube using 180 μl 1 × PBS, 20% bleach (see Additional file 4) then vortexed for no more than 30 seconds. PBS (1 ml) was added, the tube shaken then centrifuged immediately (2000 × g, 1 min). The supernatant was removed with a pipette then 180 μl 1 × PBS, 1% NP-40 was added to the pellet followed by 20 μl 32% paraformaldehyde and 2 volumes heptane (~400 μl). The sample was then processed as described above resulting in embryos in PBST-BSA.
RNase treatment (optional)
When DNA was to be stained with propidium iodide the samples were treated with RNase prior to antibody incubations. The tubes containing samples in PBST were centrifuged (2000 × g, 1 min), the supernatant removed, and replaced by 1 ml 10 mg/ml RNaseA (Sigma Aldrich, Catalog No. R4875) in PBST. The sample was rotated overnight at 4°C.
[For subsequent analysis using a fluorescence microscope or a confocal equipped with a UV laser, DNA can alternatively be stained at the end of the procedures with DAPI-containing mounting medium.]
Staining and Immunostaining
Samples were spun (2000 × g, 1 min) to remove PBST (or PBST-RNase), and resuspended in 500 μl fresh PBST. Primary antibodies were diluted into the sample, which was then rotated overnight at 4°C. Next day, the samples were centrifuged (2000 × g, 1 min), the supernatant removed and replaced by 1.5 ml PBST and the tube rotated at room temperature for > 15 minutes. This step was repeated twice but the sample resuspended in 500 μl PBST after the final wash. Secondary fluorochrome-conjugated antibodies were added according to the manufacturer's recommended dilution and the samples rotated overnight at 4°C. The tubes were centrifuged (2000 × g, 1 min), the supernatant discarded and 1.5 ml PBST added. The tubes were rotated for > 15 minutes then centrifuged again. The supernatant was discarded, 1.5 ml PBST added and, if required, 20 μl of 1 mg/ml propidium iodide (Invitrogen) added. The tube contents were mixed by shaking for 20 seconds, then centrifuged again. The supernatant was removed and replaced by 1.5 ml PBST. The tube was again shaken for 20 seconds, centrifuged, and as much liquid as possible carefully removed using a pipette. About 30 μl mounting medium (e.g. Vectashield, Vector Laboratories, Burlingame, CA, USA) was added to the tube. The mounting medium can contain DAPI if the DNA was not already stained with propidium iodide. The mounting medium and sample were mixed by gently pipetting up and down with a Pasteur pipette then transferred to a microscope slide. At this point, larger adult worm fragments can be discarded or transferred to a second slide using fine tweezers. This reduces the volume between the slide and coverslip, thereby minimizing floatation of embryos during observation. Adult fragments were mounted on a second slide using 30 μl (or more) of mounting medium. Coverslips were added and for embryo preparations, downwards pressure was applied using a paper towel to absorb excess mounting medium and stabilize the embryos. Slides were sealed with transparent nail polish and stored at 4°C in the dark. When DAPI was used in the mounting medium, the slides were stored for > 24 hr prior to microscopy to allow the stain to penetrate embryos and tissues completely.
Confocal microscope images were captured on an inverted photoscope (DMIRB; Leica Microsystems, Wetzlar, Germany) equipped with a laser confocal imaging system (TCS SP2; Leica) using an HCX PL APO 1.4 NA 63 oil objective (Leica) at room temperature.
Microtubule stainings were performed using the monoclonal DM1α antibody raised against α-tubulin (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:100. A Cy5 goat anti-mouse secondary antibody (Invitrogen) was used at 1:250. Actin stainings were performed using the fluorescent Atto 488 phalloidin (Sigma) at a dilution of 1:100.