Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

Summary

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

Abstract

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H₂O₂) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

Introduction

Aphids are hemipteran insects with small (1-10 mm) soft bodies. They feed on plants by sucking phloem sap with piercing mouthparts. Additionally, they rely on an obligate endosymbiotic bacterium, Buchnera aphidicola, to synthesize essential amino acids that are deficient in the phloem sap diet. Aphids have a complex life history that includes parthenogenetic viviparous reproduction during spring and summer long-day photoperiods and sexual oviparous reproduction triggered by short-day photoperiods during which they lay a limited number of overwintering eggs^1,2. In spring these eggs hatch to produce the first generation of all-female aphids (fundatrices), following many rounds of parthenogenetic reproduction until autumn. The cyclical parthenogenesis in aphids, where asexual and sexual phases alternate in the annual life cycle, has been regarded as an evolutionary novelty^1,2. In the parthenogenetic viviparous aphids, embryogenesis takes place within egg chambers of the ovarian tubules (ovarioles). By contrast, sexual oviparous embryos develop in the fertilized eggs. Apart from reproductive plasticity, aphids can display transgenerational wing polyphenism: in response to overcrowding signals and predator threats, the unwinged asexual females can viviparously produce winged offspring for long-distance migration. Publication of the genome sequence of the pea aphid Acyrthosiphon pisum-the first genome sequence for a basal hemimetabolous insect-allows further exploration of reproductive plasticity, wing polyphenism, and other features including insect-plant interactions, viral vectoring and symbiosis in aphids on a molecular basis³.

In addition to the sequenced genome, tools for characterizing gene expression and function are required for promoting the pea aphid as a mature model organism⁴. We have described robust protocols of whole-mount in situ hybridization for detecting expression of mRNA in aphid embryos^5-7. RNA interference (RNAi) via double-stranded RNA injection and feeding has been used for gene silencing in aphid nymphs and adults, but stable conditions for gene knockdown in the embryos have not yet been reported^8-10. Immunostaining, an antibody-based approach that can detect protein expression in samples before and after RNAi knockdown, has been performed on pea aphid embryos^11-13. However, increase of tissue permeability and elimination of background staining are as yet unsatisfactory using standard protocols for immunostaining in the asexual viviparous embryos of the pea aphid. For example, we found that penetration of antibody to the tissues decreased in gastrulating embryos (stages 8-10) and that embryos with morphologically identifiable limb buds (stages 13-14) were barely permeable to antibody. In addition, background staining was visualized in the asexual viviparous pea aphid embryos stained using antibody against the germline marker Vasa as well as that against the Engrailed/Invected protein expressed in the embryonic segments^12,13. Actually background staining was still clearly visible in embryos stained with the secondary antibody alone.

In order to increase permeability without damaging integrity of aphid tissues, we carefully titrated the concentration of proteinase K and determined optimal conditions for tissue digestion on aphid embryos. In order to avoid non-specific staining in the pea aphid, we searched for compounds that could effectively block embryos and suppress activity of endogenous peroxidase (POD), an enzyme employed for amplifying signals during immunostaining. A blocking reagent provided by a Digoxigenin (DIG)-based buffer set, rather the traditionally used normal goat serum (NGS)/bovine serum albumin (BSA), significantly reduced background staining. Moreover, methanol was found to inhibit the endogenous POD activity more effectively than hydrogen peroxide (H₂O₂). Details regarding these aphid-specific conditions for immunostaining on embryos will be described in the following sections.

Protocol

1. Culture of Aphids

NOTE: The laboratory strain of the parthenogenetic viviparous pea aphid A. pisum was originally collected in the central Taiwan and has been reared on host plants (the garden pea Pisum sativum or broad bean Vicia faba) under long-day photoperiod for more than 300 generations (one generation: ~10 days).

1. Germination of Seeds      
   1. Soak the seeds of host plants in tap water for 3-5 days at RT. Refill with fresh water once per day.
      NOTE: Alternatively, putting seeds of host plants straight into moist soil can induce germination as well.
   2. Grow 10 germinating seeds in a small pot (9 cm diameter x 7 cm tall) with soil in the growth chamber under photoperiod 16 hr light/8 hr dark at 20 °C.
   3. About 10 days after growth begins, transfer aphids onto plants whose height is more than 8 cm.  
2. Transfer of Aphids
   1. Keep each pot of plants within a 1 L glass beaker, transfer 8 adult aphids onto plants using a paintbrush, and then seal the beaker with an air-permeable cover such as gauze mesh to prevent aphids from escaping.
   2. Incubate aphids in a growth chamber under photoperiod 16 hr light/8 hr dark at 20 °C. Water each plant pot of plants once every day.
   3. For obtaining the next generation of aphids, reiterate steps 1.1.3-1.2.2 ten days after the primary aphid transfer.

2. Dissection and Fixation of Ovaries

1. Freshly prepare 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) as the fixation buffer.
2. Fill one well of a spot plate with PFA (about 500 µl), place the plate under a stereo microscope at low magnification, and submerge an adult aphid within the PFA for dissection.
3. Dissect ovaries by holding the head and abdomen with one set of forceps, cutting open the dorsal cuticle of the abdomen, and dragging the ovaries away from the abdominal cavity.
4. Fix three pairs of ovaries in a 1.5 ml tube containing 1 ml of PFA at RT for 20 min.
5. Decant fixation buffer with a transfer pipette (either glass or disposable) and then wash ovaries with 0.2% TritonX-100 in 1x PBS (PBST) 3 times for 10 min each. Mild shaking on a mixer/rotator (rotation angle: 60° (F60)/rotation speed: 8 RPM) is recommended for both fixation and washing.

3. Treatment with Proteinase K (PK) to Increase the Permeability of Embryonic Tissues

NOTE: PK treatment is applied to embryos from germ band extension onward (stage 11 of development). For younger embryos, this step is optional.

1. Serially dilute the stock solution of PK (10 mg/ml) with 1x PBS to the working concentration 1 µg/ml.
2. Incubate ovaries with 1 µg/ml of PK (about 500 µl) for 10 mins with mild shaking.
3. Decant PK solution and then wash the ovaries with 700 µl of Glycine (2 mg/ml) 3 times for 5 mins each.
4. Wash ovaries with 0.2% PBST twice for 10 min each.
5. Fix ovaries again with fixation buffer for 15 min at RT with mild shaking.
6. Discard supernatant and wash ovaries with 0.2% PBST twice for 10 min each.

4. Methanol Incubation for Suppressing the Endogenous peroxidase (POD) activity

NOTE: Methanol incubation is not applied to embryos subjected to Phalloidin staining or antibody epitopes that are methanol sensitive.

1. Serially dehydrate ovaries with different percentage of methanol in 0.2% PBST (v/v: 1:3, 1:1, 3:1) by incubating ovaries at each concentration of methanol solution for 10 min with mild agitation.
2. Dehydrate ovaries with 100% methanol for 1 hr at RT with mild shaking.
   NOTE: Satisfactory results of staining could still be obtained from aphid tissues that were stored in 100% methanol at -20 °C for one month.
3. Serially rehydrate ovaries with different percentage of methanol in 0.2% PBST (v/v: 3:1, 1:1, 1:3) by incubating ovaries at each concentration of methanol solution for 10 min with mild agitation.

5. Antibody Staining

1. Dilute the 10x blocking solution from the DIG-based buffer set (DIG-B) to 1x.
   NOTE: The 1x DIG-B blocking solution is more effective for reducing the staining background than the standard blocking reagent composed of 5% (v/v) normal goat serum (NGS) and 0.5% (v/v) bovine serum albumin (BSA) in 0.2% PBST.
2. Incubate the ovaries with the 1x DIG-B blocking solution for 2.5 to 4 hr at RT or O/N at 4 °C with mild shaking.
   NOTE: For three pairs of ovaries in a 1.5 ml tube, 200 µl is the minimum volume for sample blocking and antibody staining.
3. Decant supernatant and replace with fresh 1x DIG-B blocking solution containing primary antibody at appropriate dilution ratio. Stain the ovaries for 4 hr at RT or O/N at 4 °C with mild shaking.
   NOTE: For the experiment described here, use the following optimal dilutions of primary antibodies: (1) ApVas1 antibody: 1:500 for chromogenic staining, 1:50 for immunofluorescence staining; (2) anti-α tubulin antibody: 1:500; (3) 4D9 monoclonal antibody: 1:25.
4. Wash ovaries with 0.2% PBST 4 times for 15 min each.
5. Incubate ovaries with 1x DIG-B blocking solution for 1 hr at RT with mild shaking.
6. Decant supernatant and replace with fresh 1x DIG-B blocking solution containing secondary antibody at appropriate dilution ratio. Stain the ovaries for 4 hr at RT or O/N at 4 °C with mild shaking.
   1. Use the following dilution ratios of secondary antibodies: (1) For immunofluorescence staining: 1:500 for Alexa Fluor 633 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG; (2) For chromogenic staining: 1:200 for biotinylated goat anti-rabbit IgG.
      NOTE: The biotinylated secondary antibody is applied for interacting with the avidin-biotin-complex (ABC) in the substrate-based (chromogenic) detection, through which the staining signals are significantly amplified.
   2. For immunofluorescence staining, carry out staining in the dark because the secondary antibody is light sensitive.
7. Wash off secondary antibody with 0.2% PBST 4 times for 10 min each.

6. Nuclear and F-actin Staining

NOTE: This is only applied for immunofluorescence staining.

1. Stain ovaries with 0.2% PBST containing DAPI (2 ng/µl) and Phalloidin-TRITC (100 nM) for 2 hr at RT in dark.
2. Wash ovaries with 0.2% PBST 4 times for 10 min each.
3. Incubate ovaries with the mounting medium O/N at 4 °C with mild shaking.

7. Signal Development

NOTE: This is only applied for chromogenic staining.

1. Prepare 100 µl of reagent avidin-biotin-complex (ABC) for enhancing the signals by adding 1 µl of Reagent A (avidin) in 98 µl of 0.2% PBST, mixing thoroughly via gentle pipetting, and adding 1 µl of Reagent B (biotin conjugated with horseradish peroxidase), followed by immediate mixing. Incubate the mixture for 30 min at RT with mild shaking.
2. Incubate ovaries (including the disassociated egg chambers) in the mixture of reagents A and B for 30 min at RT with mild shaking.
3. Wash off the mixture of reagents A and B with 0.2% PBST 4 times for 10 min each.
4. Transfer ovaries submerged in PBST to a well on the spot plate with a plastic dropper.
5. Prepare substrate solution of 3,3'-Diaminobenzidine (DAB): dissolve one DAB tablet plus another one containing urea hydrogen peroxide in 1 ml of ddH₂O via vigorous vortexing for 1 min.
   NOTE: DAB, a precipitating substrate of peroxidase, is a popular chromogen for immunostaining. It produces a brown and insoluble precipitate after being oxidized by the peroxidase. Weak signals can be enhanced by adding nickel chloride to the substrate solution (final concentration 0.05-0.08 %).
6. Remove the PBST solution remaining in the well and refill with 100 µl of the DAB substrate solution for signal development.
7. Monitor intensity of signals under a stereo microscope at low magnification.
8. Stop reactions by removing the DAB solution, followed by refilling with 1x PBS immediately. Repeat the wash twice.
9. Transfer ovaries back to the 1.5 ml tube and then wash with 1x PBS or PBST twice for 15 min each. 
10. Incubate ovaries in a glycerol-based mounting medium (70% glycerol) O/N at 4 °C with mild shaking.

8. Mounting the Aphid Embryos

NOTE: The thickness of aphid embryos varies between stages of development. Mounting strategies are thus modified to fit early (germaria and stages 0-10), mid (stages 11-18), and late embryos (stages 19-20), which are demonstrated in Figure 2B-D. Embryonic staging followed Miura et al.¹²

1. Transfer ovaries together with mounting medium to the cell tray with a plastic dropper and observe samples under a stereo microscope at low magnification.
2. Cut the calyces associated with the lateral oviduct using insect pins and then transfer an isolated ovariole to an empty glass well with a dropper. Make up the final volume to 50-100 µl using mounting medium.
3. Transfer an ovariole onto the slide with a glass dropper and then dissect egg chambers using insect pins.
   NOTE: For embryos older than stage 6 of development, separation of egg chambers is suggested; for germaria and the first two egg chambers younger than stage 6, separation is optional.
4. Relocate a dissected egg chamber to a clean slide using a glass dropper.
5. Put a coverslip (size: 22 x 22 mm) over the dissected germaria or egg chambers (containing embryos at stages 1-10 of development) slowly to avoid bubbles.
   1. Mount egg chambers (containing embryos at stages 11-18 of development) on a slide with one-sided coverslip bridge and place another coverslip (size: 18 x 18 mm) on top of the sample.
   2. Mount egg chambers (containing embryos older than stage 19 of development) on a slide with double-sided coverslip bridge and place another coverslip (size: 18 x 18 mm) on top of the sample.
6. Fill the space beneath the top coverslip with mounting media to avoid drying the sample.
7. Mildly roll the embryo by sliding the coverslip to obtain the right orientation for observation.
8. Seal around the edge of coverslips (including the bridge coverslips) with nail polish.

9. Imaging Analysis

1. Photograph differential interference contrast (DIC) images of whole-mount embryos with a compound microscope equipped with DIC optics and a dry objective lens (10X, 20X, 40X) connected to a camera. Install the software for image transfer between the camera and computer using manufacturer’s instructions.
2. Acquire projections of the fluorescently labeled embryos with a laser-scanning confocal microscope¹⁴. Follow the manufacturer's instructions for photographing, z-stacking, and 3D projecting with imaging software.
   1. Turn the slide upside down, find the mounted sample with 10X objective, and circle the sample area with a fine oiled-based pen.
      NOTE: This makes identifying the sample easier when searching for it through objectives of a confocal microscope.
   2. Add a drop of oil on the top of the coverslip area whose opposite side is labeled as described in 9.2.1.
3. Find the focal plane at 40X oil-immersion objective then change to a 63X oil-immersion objective. Manually move the fine focus control up and down to capture the best focal plane.
   NOTE: For observing structural details of germaria and early embryos, we suggest using 40X objectives or those with higher magnification.
4. Scan the embryo in different excitation channels and obtain a z-stack image.
   NOTE: For pea aphid tissues, reduce thickness of each optical section down to 1.5 µm or less.

Results

In this study, we performed whole-mount immunostaining on embryos of asexual pea aphids (Figure 1A). These females produce offspring parthenogenetically and viviparously. These female embryos develop within egg chambers of the ovarian tubules (ovarioles) (Figure 1B and Figure 2A). Before microscopy, the dissected ovarioles are the staining targets; however, separation of egg chambers is required for observation of embryos under a microsco...

Discussion

We identified optimal conditions critical to successful immunostaining in the pea aphid A. pisum, an emerging model organism for genomic and developmental studies^3,15. Optimized conditions for increasing tissue permeability and reducing background staining enhanced the intensity and specificity of signals. They differ from standard protocols for immunostaining in other animal models in the steps for creating pores in cell membranes and blocking non-specific antibody binding.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30% hydrogen perxidase (H2O2)	Sigma-Aldrich	18304	For bleaching endogenous peroxidase of embryos.
4’6-diamidino-2-phenyl-indole dihydrochloride (DAPI)	Sigma-Aldrich	D9542	For labeling the double-strand DNA. TOXIC. Wear gloves, dispense into small aliquots and feeze to minimize exposure
4D9 anti-engrailed/invected mouse monoclonal antibody	Developmental studies hybridoma bank	AB_528224	For labeling the developing segments of embryo.
ApVas1 rabbit polyclonal antibody	Our laboratory	N/A	For labeling the aphid germline specific Vas1 protein in embryos. This antibody was made by our laboratory.
Austerlitz Insect pins	ENTOMORAVIA	N/A	For seperating each egg chambers from an ovariole. Size 000, BLACK ENAMELLED.
Bovine serum albumin (BSA)	Sigma-Aldrich	9048-46-8	For blocking the non-specific binding of antibodies.
Camera connted to compound microscope	Canon	EOS 5D	For photographing of aphid embryos.
Colorimetric 8 cell tray	Kartell Labware	357	For developing the staining signal of aphid embryos. Diameter 95 x 57 mm, cell depth 2 mm.
Compound microscope with DIC optics	Leica Microsystems	DMR	For photographing of aphid embryo mounted on the slides.
DIG Wash and Block Buffer Set	Sigma-Aldrich (Roche)	11585762001	For blocking the non-specific binding of antibodies. We diluted the 10x Blocking solution into 1x as blocking reagent.
Forceps	Ideal-tek	N/A	For dissection of ovaries from aphids. Manufacturer part No 5: 5 SA.
Glass dropper	N/A	N/A	For transfering ovaries of aphids from the splot plate to tubes. 150 mm of total length and 5 3/4" of tip length.
Glycerol	Sigma-Aldrich	G5516	For clearing of aphid embryos and mounting.
Glycine	Bioshop	N/A	For blocking the enzyme activity of proteinase K (PK).
Goat anti-mouse IgG conjugated Alexa Fluor 488	Invitrogen	A11017	For detection of primary antibody from mouse.
Goat anti-rabbit IgG conjugated Alexa Fluor 633	Invitrogen	A21072	For detection of primary antibody from rabbit.
Intelli mixer	ELMI laboratory equipment	RM-2M	For improving the thorughly reaction of reagents with ovaries.
Laser-scanning confocal microscopy	Leica Microsystems	SP5	For photographing of aphid embryo mounted on the slides with florecent tag.
methanol	Burdick and Jackson	AH2304	For bleaching endogenous peroxidase of embryos.
Microscope slides	Thermo scientific	10143560	For mounting of embryos. SUPERFROST ground edges, ca./env. 76 x 26 mm.
Microscope Cover glasses	Marienfeld	0101030	For mounting embryos on the slides. Size 18 x 18 mm. Thickness No. 1 (0.13 to 0.16 mm)
Microscope Cover glasses	Marienfeld	0101050	For mounting embryos on the slides. Size 22 x 22 mm. Thickness No. 1 (0.13 to 0.16 mm)
mouse monoclonal anti-alphaTubulin antibody (DM1A)	Santa Cruz Biotechnology	sc-32293	For labeling the distrbution of alpha-tubulin of cells.
Nail polish	N/A	N/A	For sealing the coverslips on the slides.
Normal goat serum (NGS)	Sigma-Aldrich	G9023	For blocking the non-specific binding of antibodies.
Paraformaldehyde (PFA)	VWR	MK262159	For fixation of aphid ovaries.
Phalloidin-TRITC	Sigma-Aldrich	P1951	For labeling the distrbution of F-actin on embryos.
Phosphate-buffered saline (PBS)	N/A	N/A	As isotonic solution for aphid ovaries.
Plastic dropper	N/A	N/A	For transfering ovaries of aphids from tube to cell tray. Size 3 ml.
Proteinase K	Merck	124678	For creating pores (punching) on the cell membrane and facilitating the access of antibodies.
Pyrex spot plate	N/A	N/A	For dissection and color reactions of aphid ovaries under microscopy. Each cavity with diameter 22 mm and  depth 7 mm.
SIGMAFAST 3,3’-diaminobenzidine (DAB) tablets	Sigma-Aldrich	D4168	For developing of substrated signals. Also called DAB peroxidase substrate tablet set.
Stereo microscope	Leica Microsystems	EZ4	For dissection and observation of aphid ovaries.
Triton-X 100	Sigma-Aldrich	T8787	For creating pores (punching) on the cell membrane.
VECTASHIELD Elite ABC Kit (Rabbit IgG)	Vector laboratories	PK-6101	For enhancement of the signal. Including of biotinylated goat anti-rabbit IgG secondary antibody, A and B reagents.
VECTASHIELD mounting medium	Vector laboratories	H1000	For clearing of aphid embryos and mounting.