Lepidopteran fat body whole-mount immunofluorescence (Nymphalidae)

Knowledge graph centered on Lepidopteran fat body whole-mount immunofluorescence (Nymphalidae) with 16 nodes and 47 connections. Top connected: Bombus, Bombus terrestris, Lepidoptera, early-life trade-offs, Speyeria mormonia.

Method synopsis

A whole-mount immunostaining protocol optimized for visualizing FOXO and mTOR protein localization in butterfly fat body tissue using species-specific antibodies. The protocol involves tissue fixation, permeabilization with detergents, blocking, and fluorescent antibody labeling.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — Antibody development to identify components of IIS and mTOR signaling pathways in lepidopteran species, a set of non-model insects (2023), microPublication Biology. Implementations in other papers (listed below) may differ.

1. Specimen sacrifice and dissection
   Females were sacrificed by crushing the ventral nerve and their abdomens were immediately dissected in cold phosphate buffered saline (PBS) to obtain fat body. Fat bodies were placed in 1.5ml microfuge tubes that were previously coated with 3% bovine serum albumin (BSA).
   Quantities: Individual fat body tissues from multiple femalesDuration: Immediate processing after sacrificeConditions: Cold PBS during dissection
   Equipment: 1.5ml microfuge tubes, BSA coating
2. Tissue fixation
   Fat bodies were fixed in 1ml of 4% paraformaldehyde diluted in cold PBS (PFA) for 30 minutes at room temperature, rocking on a nutator.
   Quantities: 1ml of 4% PFA per sampleDuration: 30 minutesConditions: Room temperature with rocking motion
   Equipment: nutator, 4% paraformaldehyde
3. Washing and permeabilization
   The PFA was removed, and samples were rinsed twice with 1ml of PBS containing either 0.1% Triton X-100 (PBTX) or Tween 20 (PBTW) as a detergent. After rinsing, samples were washed three times with 1ml of 0.1% PBTX or PBTW, rocking each time for 15min on a nutator at room temperature.
   Quantities: 1ml wash volume per rinse and wash step

Typical Equipment

• 4% paraformaldehyde
• Triton X-100
• Tween 20
• BSA
• Rabbit anti-FOXO antibody
• Rabbit anti-TOR antibody
• Goat anti-rabbit AlexaFluor 488
• VectaShield with DAPI
• nutator

Output Measurements

• protein subcellular localization
• immunofluorescence intensity
• tissue-specific protein distribution

Papers Using This Protocol (1)

Species Studied With This Protocol