Laser microdissection combined with immunohistochemistry on serial thin tissue sections: a method allowing efficient mRNA analysis

Abstract

Laser microdissection (LMD) with subsequent reverse transcription-PCR analysis is a powerful histochemical technique subserving the molecular characterization of specific cell types. We developed an efficient method for selective sampling of specific cell populations using immunohistochemistry coupled with LMD. The cerebral cortex of adult rats was cut into serial thin sections. Some sections were immunostained for parvalbumin. The adjacent sections were mounted on Cell Support Film for LMD and stained with neutral red. By comparison of the two adjacent sections, neuronal profiles representing parts of parvalbumin-immunopositive somata were identified in the neutral red-stained sections. These neuronal profiles were safely captured with LMD and analyzed on reverse transcription-PCR using extracted RNA. The method presented here can be applied to cell-type-specific characterizations using fixed cells under RNase-free conditions.

Histological Determination of the Areas Enriched in Cholinergic Terminals and m2 and m3 Muscarinic Receptors in the Mouse Central Auditory System

“…The brain was sliced into 40‐μm coronal sections with Cryotome according to Kase et al (2007). These sections were immunostained for VAChT with essentially the same method as described previously (Tsutsumi et al, 2007).…”

State-of-the-art technologies, current opinions and developments, and novel findings: news from the field of histochemistry and cell biology

“…Image-assisted RNA and protein analysis: molecular proWling in tissues and cells Laser microdissection has been in use to determine RNA expression in individual cells for some time, and methods have been described to identify and characterize the analyzed cells prior to or concurrent with the RNA analysis (e.g., Kase et al 2007). Two exciting recent reviews report advances in the analysis of cellular RNA and protein proWles which represent giant steps towards the application of these sophisticated techniques to basic and clinical research.…”

Methods and Models for Monitoring UPR-Associated Macrophage Death During Advanced Atherosclerosis

“…In parallel analyses, we have confirmed that the majority of directly adjacent sections exhibit equivalent immunostaining for F4/80+ regions of interest when cut at 6 μm. A similar approach has been applied for LCM combined with IHC in brain (Kase et al , 2007). For parallel LCM slides, perform rapid staining as described by Ambion (AM1935 LCM Staining Kit) to provide contrast and for alignment with the identified regions of interest.…”