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In Situ Hybridization (view source)
Revision as of 14:45, 22 November 2011
, 14:45, 22 November 2011added methods and reagent ordering info
=Interpretation of ISH results=
In situ hybridization visualizes mRNA expression in intact tissue sections thus providing anatomical localization. Because it employs DNA or RNA probes complimentary to specific mRNA sequences, in situ hybridization visualizes only the product of the target gene and not related gene products; it thus can provide greater specificity than immunohistochemistry, which may visualize proteins that have a common epitope but are otherwise unrelated to the target protein.
A significant advantage of doing in situ hybridization on free-floating tissue sections (as opposed to slide-mounted sections) is that sections from different rats may be hybridized at the same time within the same vial, and thus under identical conditions. Tissue sections from different rats are distinguished by labeling the sections during cutting with notches or punctures, so that they may be sorted by individual after hybridization. Because rats from control and experimental groups are processed under identical conditions, quantitative comparisons are more reliable.
==Protocol==
Herring Sperm DNA serves as blocker of nonspecific DNA binding
DTT (dithiothreitol) (aka Cleland's reagent) prevents oxidation of thiol groups and breaks down disulfide bonds
==Reagents and Solutions==
''prices as of 2011-11''
DEPC (diethylpyrocarbonate), Sigma #D-5758-50 ml $203
Dextran Sulfate sodium salt, Sigma #D-8906-50 g, $143
20x SSC Buffer 20x Ultrapure, 1L bottle, Invitrogen #15557-044 $34
Denhardt's Solution 50x, 100 ml, Invitrogen # 750018 $122
:(made from PVP (polyvinyl pyrrolidone) 40 & Ficoll Type 400)
Salmon Sperm DNA 10 mg/ml, 10 x 1 ml tubes, Invitrogen #AM9680 $198
DTT (dithiothreitol) (aka Cleland's reagent) Ultrapure, 5 g, Invitrogen # 15508013 $102.00
==Methods Section==
===cDNA ISH===
Reference:
[preparation of cDNA probes from restriction fragments or PCR products, using random-priming reaction, e.g. HiPrime?]
Free-floating tissue sections were collected into 20 ml glass scintillation vials containing ice-cold 2 X SSC (0.3M NaCl, 0.03M sodium citrate) for in situ hybridization. The SSC in each vial was pipetted off, and sections were then suspended in 1 ml of warm prehybridization buffer (50% formamide, 10% dextran sulfate, 2 X SSC, 1X Denhardt’s solution, 50mM dithiothreitol, 0.5 mg/mL denatured salmon sperm DNA) and placed in a 48°C water bath. Two h later, 35S-dCTP-labeled cDNA probes (10 x 10^6 CPM/vial) were added to the vials and hybridized overnight at 48°C. After overnight hybridization, the sections were washed at 15-min intervals in decreasing concentrations of SSC (2X, 2X, 1X, 0.5X, 0.25X, 0.125X, 0.125X) at 48°C. After washes, the tissue sections were stored in 0.1M phosphate buffer at 4°C and then mounted on gelatin-subbed slides, air-dried, and apposed to Kodak Biomax autoradiographic film (Eastman Kodak Co., NY,
===Riboprobe ISH===
Reference: Kwon, B.S., M. Goltz, and T.A. Houpt. Expression of AP-1 family transcription factors in the amygdala during conditioned taste aversion learning: role for Fra-2. Brain Res. 1207 (2008) 128-41. PMID 18374904. PMCID PMC2756721.
For in situ hybridization, antisense RNA probes of [c-fos, fra-2, c-jun, and junD] cDNAs were made by in vitro transcription. Amplified cDNAs from RT-PCR were inserted into pCRII-TOPO cloning vectors (Invitrogen, Carlsbad, CA). The cloning vectors containing each cDNA were linearized by restriction enzymes. The linearized plasmids were purified by QIAquick spin column (Qiagen, Valencia, CA), and the MAXIscript kit (Ambion, Austin, TX) was used for in vitro transcription. The linearized plasmid template (1 µg), 2µl of 10X transcription buffer, 1µlof ATP, CTP, and GTP (each 10 mM), 5µl of 35S-labeled UTP (20 mci/ml) (Amersham, UK) 2µl of T7 or SP6 RNA polymerase, and RNase-free H20 were mixed in a total reaction volume of 20 µl. The mixture was incubated at 37 ºC; after 30 min, DNase I (1 µl) was added and the incubation continued for another 15 min. After stopping the reaction by the addition of EDTA (1 µl, 500 mM), the probe solution was purified through ProbeQuantTM G-50 spin columns (Amersham, Piscataway, NJ).
Forty micron free floating sections were cut on a freezing, sliding microtome and transferred into 20-ml glass scintillation vials containing 0.15 M NaCl, 0.015 M sodium citrate (2xSSC) buffer. Sections were prehybridization at 55ºC for 2-3 h in 1 ml per vial of hyrbidization buffer (50% formamide, 2xSSC, 10% dextran sulfate, 0.7% Ficoll, 0.7% polyvinylpyrrolidone, 0.7% bovine serum albumin (BSA), 85 mM dithiothreitol (DTT) and 1.4 mg/ml of yeast transfer RNA). Sections were hybridized at 55ºC for 18 h with heat-denatured 35S-labeled or biotinylated antisense RNA probes. Sections were then washed sequentially in 2xSSC, 2xSSC, 1xSSC, 0.5xSSC, 0.25xSSC, 0.125xSSC, 0.125xSSC at 55ºC for 15 min each. To reduce background, tissue sections were incubated with RNase A (30 ug/ml) both before and after mounting on gelatin coated slides. Finally, slides were washed sequentially in 2xSSC/50% formamide/0.1% β-mercaptoethanol at 53ºC for 15 min, 0.1xSSC/1% β-mercaptoethanol at 53ºC for 30 min, 50% ethanol/0.3 M NH4OAC at RT for 3 min, 85% ethanol/0.3 M NH4OAC at RT for 3 min, 100% ethanol at RT for 3 min.
Radiolabeled tissue sections on slides were apposed to Biomax MR film. Biotin-labeled slides were processed for immunohistochemistry with anti-biotin antiserum (Roche) and fluorescently-tagged secondary antibodies (Vector) for visualization by fluorescent microscopy.
===Oligo ISH===
===Quantification of ISH===
Reference:
For ISH results, pixel density is quantitated from the autoradiographic films using a custom software program (MindsEye, T. Houpt). Films were digitized through a Zeiss Stemi2000C dissecting scope with a Fostec flat fiberoptic light source; light levels are adjusted to standardize gray levels of film background across ISH experiments. Images werw captured in a 10 mm x 7.5 mm frame using a scientific-grade CCD camera (Dage MTI DC330E) [UPDATE CAMERA] and frame-capture board (Scion CG-7). The region of interest (i.e. the arcuate nucleus) is delineated automatically by an algorithm that finds clusters of 10 or more contiguous pixels with an intensity 2 standard deviations above the average tissue background (mRNA-positive pixels). This algorithm works particularly well on structures with relatively discrete patterns of gene expression. Arbitrary units of mRNA expression are then derived by summing the intensities of all the mRNA-positive pixels, and subtracting the average tissue background value. For each rat and probe, average pixel densities were obtained from 3-5 brain sections. Individual mean values for each region were then averaged across rats within experimental groups.
==Interpretation of ISH results==
'''Changes in area vs. density of autoradiographic in situ hybridization signal'''
'''Changes in area vs. density of autoradiographic in situ hybridization signal'''