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Line 5: − 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''+ + + + + + + Line 18:
Line 23: − 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)+ − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Line 33:
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Follow redirect from Perkin to new Revvity
==Protocol==
==Protocol==
==Reagents==
''prices as of 2011-11''
AG 501-X8 Resin (Mixed bed resin for deionizing formamide), 100g, BioRad #143-7424, $323
[https://www.revvity.com/product/datp-a-thio-35s-neg034h250uc dATP-alphaS35], (250 uCi vial = 20 ul) [https://www.perkinelmer.com/corporate/contactus Perkin Elmer] #NEG034H250UC $172
:made first Tuesday of every month
Denhardt's Solution 50x, 100 ml, Invitrogen # 750018 $122
:(made from PVP (polyvinyl pyrrolidone) 40 & Ficoll Type 400)
DEPC (diethylpyrocarbonate), Sigma #D-5758-50 ml $203
DEPC (diethylpyrocarbonate), Sigma #D-5758-50 ml $203
Dextran Sulfate sodium salt, Sigma #D-8906-50 g, $143
Dextran Sulfate sodium salt, Sigma #D-8906-50 g, $143
DTT (dithiothreitol) (aka Cleland's reagent) Ultrapure, 5 g, Invitrogen #15508013 $102.00
Formamide Ultrapure, 500g bottle, Invitrogen #15515026 $97
'''For cDNA ISH:''' High Prime DNA Labeling Kit, 50 reactions, Roche Applied Science, #11585584001 $333
'''For Oligo ISH:''' Terminal Transferase Tail-Labeling Kit,
ProbeQuant G-50 micro spin columns, 50 pack, GE Healthcare, # 28-9034-08 $199
Salmon Sperm DNA 10 mg/ml, 10 x 1 ml tubes, Invitrogen #AM9680 $198
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
SSC Buffer 20x Ultrapure, 1L bottle, Invitrogen #15557-044 $34
== Solutions==
'''DEPC-Treated Water'''
*1 ml of DEPC per 1 L ddH2O
*Squirt DEPC forcefully into water.
*Cover and shake vigorously.
*(Some protocols recommend sitting overnight.)
*Autoclave.
'''20x SSC'''
''(We now purchase this from Invitrogen).
*400 ml DEPC-H2O
*87.65 g NaCl
*441 g Citric Acid
*Bring to pH 7.0
*Bring volume to 500 ml with DEPC-H2O
*Autoclave. (make sure volume back to 500 ml)
Hybridization of DNA or RNA probes to target RNA is dependent on salt concentration (Na+ neutralizes the PO3- charges on the DNA/RNA backbones.) SSC is an old-time decoagulant that was a common buffer in early biochemical labs. The hybridization conditions are optimized for 2x SSC.
'''Deionized Formamide:'''
*Mix 50 ml formamide and 5 g of mixed bed ion exchange resion (Bio-Rad AG 501-X8, 20-50 mesh).
*Stir 30 min at room temperature.
*Filter twice thru Whatman #1 filter paper
*Store at -20C in 15 ml tubes covered with aluminum foil.
Formamide denatures nucleic acids (lowers effective Tm). Because the breakdown products of formamide degrade nucleic acids, for sensitive applications formamide should be deionized by treatment with a mixed-bed ion-exchange resin.
'''5x TED'''
*250 mM Tris, pH 7.4, 25 mM EDTA, 50% Dextran Sulphate
*Weigh 3.05 g Trizma Base
*Weigh 0.93 g EDTA
*Add ~35-40 ml DEPC-H2O
*Bring to pH 7.4 with HCL
*Add 50 g Deteran Sulfate slowly, while heating slowly to boil
*Bring volume to 100 ml with DEPC-H2O
*Store at 4C in 15 ml tubes covered with aluminum foil
Dextran is corn starch; serves as a thickener to increase effective concentration of all the other reagents.
'''50x Denhardt's Solution'''
''(We now purchase this from Invitrogen).
*1% Ficoll, 1% PVP, 1% BSA
*1 g Ficoll, 1 g PVP, 1 g BSA in 100 ml DEPC-H2O.
*Filter.
*Store in 1 ml tubes at -20C.
Denhardt's is a mixture of high molecular weight polymers that blocks non-specific binding sites
'''Herring/Salmon Sperm DNA'''
''(We now purchase this from Invitrogen).
*Dissolve 10 mg salmon or herring sperm DNA per 1 ml DEPC-H2O
*Extraction with phenol and phenol-chloroform optional.
*Sonicate for a few minutes until solution is no longer viscous (~3 min)
*An OD260 to determine exact concentration of DNA is also optional.
*Boil solution for 10 min
*Cool on ice
*Aliquot at 800 ul and store at -20C
Random DNA serves as blocker of nonspecific DNA binding
'''DTT (dithiothreitol)'''
Aka Cleland's reagent; prevents oxidation of thiol groups and breaks down disulfide bonds.
==Buffer Amounts==
* Denature salmon sperm by boiling for 10 minutes.
* Put DTT in foil-covered scintillation vial.
* Add SSC, formamide, TED, salmon sperm, & Denharts solution.
* Vortex. Keep heated at 55° C on heater block until use.
<tab class="wikitable" sep="tab" border = "1" cellspacing="3" cellpadding = "3" head = top>
10 ml - - - 8 ml - - - 5 ml - - - 4 ml - - - 3 ml - - - 2 ml - -
150 mg DTT 120 mg DTT 75 mg DTT 60 mg DTT 45 mg DTT 30 mg DTT
1.0 ml SSC 20x 800 ml SSC 20x 500 µl SSC 20x 400 µl SSC 20x 300 µl SSC 20x 200 µl SSC 20x
6.0 ml Formamide 4.8 ml Formamide 3.0 ml Formamide 2.4 ml Formamide 1.8 ml Formamide 1.2 ml Formamide
2.0 ml TED 5x 1.6 ml TED 5x 1 ml TED 5x 800 µl TED 5x 600 µl TED 5x 400 µl TED 5x
1.6 ml Salmon Sperm 1.28 ml Salmon Sperm 800 µl Salmon Sperm 640 µl Salmon Sperm 480 µl Salmon Sperm 320 µl Salmon Sperm
800 µl Denharts 50x 640 µl Denharts 50x 400 µl Denharts 50x 320 µl Denharts 50x 240 µl Denharts 50x 160 µl Denharts 50x
</tab>
===cDNA ISH===
===cDNA ISH===
Reference:
Reference: From Rivera et al, 2009, PMID 19168037
[preparation of cDNA probes from restriction fragments or PCR products, using random-priming reaction, e.g. HiPrime?]
Prepare cDNA probes by [[cDNA Random Priming]].
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,
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,
===Oligo ISH===
===Oligo ISH===
Reference: Swart, I., J.M. Overton, and T.A. Houpt. Hypothalamic NPY, AGRP and POMC mRNA responses to leptin and refeeding in mice. Am. J. Physiol. 283 (2002) R1020-R1026. PMID 12376393.
Prepare probes by [[Oligo Tail Labeling]].
Coronal 40 micron sections were cut on a sliding freezing microtome through the rostral caudal extent of the hypothalamus. Alternate sections were placed into ice-cold 2x SSC (SSC = 0.15M NaCl/0.015M Na Citrate). Free floating tissue sections were prehybridized in glass vials in 1ml of 60% formamide, 0.02 M Tris pH7.4, 1mM EDTA, 10% dextran sulfate, 0.8% Ficoll, 0.8% PVP, 0.8% BSA, 2x SSC, 0.1M dithiothreitol, and 1.6 mg/mL herring sperm DNA for 2h at 37°C. After 2h prehybridization, radiolabeled probe was added (~1.0 x 107 cpm/vial) and incubated for 16-20h at 37°C. Hybridization was performed with either tail labeled prepro NPY (ppNPY) antisense oligonucleotide (bases 59-88), agouti-related protein (AGRP) antisense oligonucleotide (bases 1-45) or proopiomelanocortin (POMC) antisense oligonucleotide (bases 482-517). The oligonucleotides were tail-labeled to approximately equal specific activities (~10^7 cpm/100ng) with 35S-dATP by terminal transferase reaction (Roche), Sections were then sequentially rinsed in 2x SSC, 1x SSC and 0.5x SSC for 10 minutes at 37°C. The tissue sections were mounted from 0.05M sodium phosphate onto gelatin- coated slides, air dried, and exposed to autoradiographic film (β-max, Amersham) for 2-3 days. Tissue sections from different groups were hybridized in parallel and exposed to film together to ensure that in situ hybridization was carried out on representative members of each experimental group at the same time under identical conditions, allowing direct comparison of mRNA expression.
===Quantification of ISH===
===Quantification of ISH===
Note that the ceiling or maximal level of expression may vary across regions, even for the same gene product. For example, the MR has a lower and sparser expression of 5HTT and Pet-1 mRNA than the DR under baseline conditions. Thus the dynamic range of MR cells may be lower than cells in the DR, and an increase in expression within the MR may be detected as an increase in area rather than in density. The physiological bases for the heterogenity in baseline and stimulated levels of gene expression within the raphe nuclei is a profound topic, to which this paper makes a small contribution with respect to estrogen effects.
Note that the ceiling or maximal level of expression may vary across regions, even for the same gene product. For example, the MR has a lower and sparser expression of 5HTT and Pet-1 mRNA than the DR under baseline conditions. Thus the dynamic range of MR cells may be lower than cells in the DR, and an increase in expression within the MR may be detected as an increase in area rather than in density. The physiological bases for the heterogenity in baseline and stimulated levels of gene expression within the raphe nuclei is a profound topic, to which this paper makes a small contribution with respect to estrogen effects.
[[Category:Protocols]]