In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Summary

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

Abstract

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes. In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.

Protocol

DAY 1

1. Prepare the following solutions:
   • 10x PBS
   • 1x PBS
   • 1x PBT
   • ddH₂O
     • Add DEPC for 0.1% total volume and shake solutions vigorously. 
     • Autoclave.
   • 4% Paraformaldehyde Fix - using PBS-DEPC
   • Proteinase K solution - if precipitate is present, vortex vigorously before removing aliquot
   • Digestion Stop Buffer
   • Prehybridization Buffer
   • 50:50 PBT:Prehybridization Buffer
   • Hybridization Buffer
     • Solutions should be kept RNase-free. Use either glass-ware that has been baked (4 hours at 250°C) or disposable plastic. Serological pipettes are very useful for making up solutions
2. Dissect wings from time-staged pupae in PBS
3. Move wings directly to Fix Buffer in wells of 24 well culture plate at room temperature
4. Fix discs for 2 hours
5. Wash 5 x 5 minutes in PBT
6. Incubate wings for 3 minutes in Proteinase K solution
7. Rinse 2 x in Digestion Stop Buffer
8. Wash 5 x 5 minutes in PBT
9. Wash 2 x 5 minutes in 50:50 PBT : PHB
10. Wash 1 x 10 minutes in PHB
11. Incubate in PHB at least 1 hour at 55°C.
12. Heat-denature RNA probe (20-50ng needed per well) (80°C for 5 minutes) and add to hybridization buffer (100 μl needed per well)
13. Add probe to wells and incubate 48 hours at 55°C 

DAY 3

1. Wash 4 x 5 minutes in 55°C PHB
2. Incubate overnight in PHB at 55°C

DAY 4

1. Prepare the following solutions:
   • Antibody Block Buffer
2. Wash 1 x 5 minutes in 50:50 PBT : PHB
3. Wash 4 x 5 minutes in PBT
4. Incubate wings for 1 hour in Block Buffer at 4°C
5. Incubate wings in a 1:2000 dilution of anti-Dig antibody overnight at 4°C

DAY 5

1. Prepare following solutions:
   • Detection Buffer
   • Developing Solution - light sensitive (wrap in foil)
2. Wash 3 x 5 minutes in PBT at room temperature
3. Wash 7 x 5 minutes in PBT
4. Rinse wings two times in Detection Buffer
5. Develop wings in 1 ml of Developing Solution - time of development must be monitored each time - developing times can change even when using the same probes and developing solution - generally check after 5-10 minutes and then increase intervals up to overnight. Plate should be wrapped in foil to prevent light exposure when not checking samples.
6. Rinse five times in Developing Stop Buffer
7. Mount wings.

Discussion

Optimization of existing protocols

In situ hybridizations on larval wing discs have been successfully performed using discs from Precis coenia butterflies (Carroll et al. 1994; Keys et al. 1999; Weatherbee et al. 1999). The current protocol has been adapted from a detailed written protocol available upon request from the Carroll Lab for larval wing stainings. The changes we made serve to accommodate differences between larval and pupal wing tissues. During pupal hindwing dissections, the p...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fix Buffer				4% Paraformaldehyde in PBS
PBT				0.1% Tween 20 in PBS
Proteinase K solution				2.5mg/ml Proteinase K in PBT
Digestion Stop Buffer				2 mg/ml glycine in PBT
Pre-Hybridization Buffer				For 50 ml PHB: 12 ml DEPC treated water + 25 ml Formamide + 12.5 ml 20 x SSC + 50 ul Tween 20 + 500 ul 10 mg/ml salmon sperm (Rnase free, heat denatured prior to addition to solution).
Hybridization Buffer				Add 1 mg/ml glycogen to prehybridization buffer
Block Buffer				50 mM Tris pH 6.8, 150 mM NaCl, 0.5% IGEPAL (NP40), 5 mg/ml BSA
Anti-DIG	Ab	Roche Group	11 093 274 910	Alkaline Phosphatase conjugated
DIG Wash and Block Buffer Set		Roche Group	11 585 762 001
Crystal Mount Aqueous Mounting Medium		Sigma-Aldrich	C0612	Mounting Medium