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Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.
Until today, iron gall ink is classified as an exceptional underdrawing material for paintings. Its study and definite identification is usually based on invasive analysis. This article presents a new non-destructive approach using micro-X-ray fluorescence scanning (MA-XRF), LED-excited IRR (LEDE-IRR) based on a narrow wavelength-range of infrared radiation (IR) for illumination and stereomicroscopy for studying and visualising iron gall ink underdrawings. To assess possibilities and limits of these analytical techniques, the approach was tested on panel paintings by Hans Holbein the Elder and Giovanni Battista Cima da Conegliano. Results are compared to invasive examinations on cross-sections using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX). The holistic setup could successfully visualise iron gall ink underdrawings, allowing to harness the formerly invisible underdrawing lines for interdisciplinary studies.
Highlights
• Protocol for extracting and analyzing pollen grains from fossil insects
• Individual fossil grains can be analyzed using a combined approach
• Simple and fast TEM embedding and sectioning protocol
• Protocol enables a taxonomic assignment of pollen
Summary
This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison.