HistoGene® LCM Immunofluorescence Staining Kit

• Simplify Fluorescent Target Cell Labeling
• Use a Quick 15-minute Process to Preserve mRNA
• Chill Samples for Intact RNA
• Get Brilliant High-contrast Label Intensity
• Obtain Intact RNA from Many Tissue Types
• Use Microgenomic Products for Microarray Analysis

Simplify Fluorescent Target Cell Labeling

Identify target cells for Laser Capture Microdissection (LCM) and gene expression analysis using antigen markers to highlight specific surface or intracellular proteins. The HistoGene™ LCM Immunofluorescence Staining Kit is the only Kit specifically designed for retrieval of high-quality RNA from immunofluorescently stained frozen tissue. The Staining Kit provides reagents and slides for convenient and reliable immuno-flourescent staining. The Kit provides protocols streamlined and optimized to maintain RNA quality and materials sufficient for processing 32 slides.

Laser Capture Microdissection (LCM) of human prostate ductal epithelium prepared with the HistoGene LCM Immuno-fluorescence Kits. Panel A. Human prostate stained with an anti-cytokeratin antibody, before LCM. Panel B. Image of the captured cytokeratin positive cells on the cap after LCM.

Use a Quick 15-minute Process to Preserve RNA

Standard immunofluorescent labeling kits generally take over 90 minutes to process samples, compromising the RNA integrity with conditions sub-optimal for single-stranded nucleic acid stability. In contrast, the HistoGene LCM Immunofluorescence Kits' protocols take less than 15 minutes (only 5 minutes in aqueous environment) and are performed using Arcturus' proprietary staining buffer, which minimizes RNA degradation. Electrophoresis and RT-PCR of RNA isolated after using the HistoGene Immuno-fluorescence Kits confirm that high-quality RNA is retrieved from microdissected samples.

Comparison of RNA quality before and after processing using the HistoGene LCM Immunofluorescence Kits. RNA was prepared from unstained but dehydrated frozen section of mouse small intestine (Lane 1), and from frozen section of small intestine stained using the HistoGene LCM Immunofluorescence Kit to visualize vascular expression of MAdCAM-1 (Lane 2). Electrophoretic analysis of total cellular RNA isolated from mouse small intestine was performed on an Agilent Bioanalyzer 2100.

Detection of low- and high-abundance, full-length RNA in HistoGene LCM immunofluorescent stained samples. RT-PCR was performed on RNA from 500-1000 cells captured from different tissues. Equal quantities of cDNA were analyzed with primers detecting 3' and 5 ' ends of low-abundance ADP ribosylation factor 1 ( ARF-F1), (3'; 239 bp, 5'; 336 bp) and 5' end clathrin gene which has a transcript length over 6 kb (570 bp) (primer sets from KPL, Inc.). M: molecular-weight markers. Lane 1: human breast tissue, ARF-F1, 5'. Lane 2: mouse brain, ARF-F1, 5'. Lane 3: mouse spleen, ARF-F1, 3'. Lane 4: mouse lacrimal gland, ARF-F1 3'. Lane 5: mouse lacrimal gland, clathrin. Lane 6: human prostate, clathrin. Lane 7: negative RT control, human prostate, clathrin.

Chill Samples for Intact RNA

To increase RNA yield and ensure its quality, the HistoGene Cold Block keeps up to four tissue section slides and several tubes containing buffer and antibody solutions chilled during HistoGene LCM Immunofluorescence Kit processing. The block is designed for use with the CoolSafe triple density polysterene cooler (Cat. # CSF-Box) and -10ºC CoolBrick (Cat. # BRIK-1520) from Diversified Biotech.

Get Brilliant High-contrast Label Intensity

The HistoGene LCM Immunofluorescence Kits employ a biotin-avidin system with Cy3 which leads to exceptionally good staining intensity and specificity. The Kit is used with a primary biotinylated monoclonal antibody to an antigen of choice provided by the user and Cy3-conjugated streptavidin is included in the Kit.

Obtain Intact RNA from Many Tissue Types

Arcturus scientists have examined RNA integrity after using the HistoGene Immunofluorescence Kits on many tissue types with several antibodies. All tissue tested using the HistoGene Immuno-fluorescence Kits have yielded high-quality RNA.

Use Microgenomic Products for Microarray Analysis

The HistoGene LCM Immunofluorescence Kits are part of Arcturus' complete Systems for Microgenomics--products designed to seamlessly work together to produce high-quality expression microarray data from pure cell populations.

Use HistoGene-stained samples with the Arcturus^XT and Veritas Microdissection Instruments to capture pure cell populations of flourescently labeled target cells. Maximize recovery of RNA from even small numbers of cells with the PicoPure RNA Isolation Kit. Next, amplify nanogram quantities of RNA to micrograms using the RiboAmp RNA Amplification Kit, which provides amplified antisense RNA ready for labeling and hybridization to microarrays.

Expression microarray of LCM samples prepared using Arcturus' HistoGene LCM Immunofluorescence, PicoPureä RNA Isolation, and RiboAmpä RNA Amplification Kits. Human breast tumor tissue sections were stained for cytokeratin using the HistoGene LCM Immunofluorescence Kit and 1000- positively stained cells were microdissected. RNA was isolated using the PicoPure RNA Isolation Kit and amplified with the RiboAmp RNA Amplification Kit. Amplified RNA was converted to Cy5 labeled cDNA and hybridized to a 24,000 element human cDNA array and scanned.