• Hybridization Histochemistry

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₂HPO₄
H₂O to 1 liter

3.7% Formaldehyde solution

Per liter, add 100 ml 37% formaldehyde and 100 ml 10X PBS

0.25% acetic anhydride, pH 8.0

Per 100 ml, mix 1.49 ml triethanolamine and 420 µl concentrated HCl. Then add and mix 0.25 ml acetic anhydride.
Use immediately.

20X SSPE

Dissolve 175.2 g NaCl, 27.6 g NaH₂PO₄*H₂O and 7.4 g Na₂EDTA in 800 ml H₂O. Adjust pH to 7.4 with NaOH and then volume to 1 liter with H₂O. Final [Na] of 20x SSPE=3.2M.

Ribonucleic acid solution

Dissolve salmon sperm DNA (100 µg/ml final; per ml: 250 µl of 10mg/ml stock), yeast total RNA (250 µg/ml final; per ml: 313 µl of 20mg/ml stock) and yeast tRNA (250 µg/ml final; per ml: 250 µl of 25mg/ml stock) in H₂O. Store indefintely at -20° C.

50X Denhardts solution

1 g ficoll
1 g polyvinylpyrrolidone
1 g bovine serum albumin
H₂O to 100ml
Store indefintely at -20° C.

Hybridization buffer

23.8 ml formamide
0.95 ml 1M Tris-HCl, pH 7.4
0.19 ml 250 mM EDTA, pH 8.0
3.75 ml 4M NaCl
9.52 ml dextran sulfate
0.95 ml 50X Denhardts solution
H₂O to 40ml.
Store indefintely at -20° C.

Hybridization solution

Add probe plus H₂O to 8 µl plus 4 µl ribonucleic acid solution and mix. Heat at 65° C for 5 min. and then cool rapidly on ice to room temperature. Add 84 µl hybridization buffer, 2 µl 5M DTT (5g DTT plus 2.72ml H₂O), 1 µl 10% sodium thiosulfate, and 1 µl 10% sodium dodecyl sulfate. Mix well. Scale up appropriately.

RNase A solution

Dissolve 1 ml of 10 mg/ml RNase A (e.g., Sigma R6513 dissolved to 10mg/ml in 10mM Tris-HCl, pH7.5/15mM NaCl. Stored at -20° C. No boiling to denature DNase is recommended) just prior to use in 500 ml pre-warmed (37° C) RNase buffer (62.5 ml of 4M NaCl, 2.5 ml of 2M Tris-HCl, pH8.0, and 0.5 ml of 0.25M EDTA)

We have had RNase A lots that are more potent than others, so when new RNase A is obtained, it should be tried over a range of concentrations to determine the best signal-to-noise ratio.

Northern hybridization solution

20 ml formamide
2 ml 20X SSPE
8 ml 50% Dextran Sulfate
4 ml 50X Denhardt's solution
400 µl 25 mg/ml tRNA
500 µl 20 mg/ml total yeast RNA
400 µl 10 mg/ml single-stranded DNA
400 µl 10% SDS
H₂O to 40 ml
Store indefintely at -20° C.

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COMMENTARY
Background Information
Hybridization histochemistry provides a method to detect specific mRNAs in tissue sections. Furthermore, as mRNA levels may change from one state to another (e.g., during development or after physiological manipulations), hybridization histochemistry can provide snapshots through the course of a dynamic situation. This unit reprises our protocols to examine the expression of genes within tissue sections at a light microscopic resolution (Young et al., 1986; Young et al., 1990; Bradley et al., 1992; Young, 1992). There are a number of excellent sources of information for the reader interested in localizing transcripts in whole mount tissues, to chromosomes or at the electron microscopic level (Wilkinson, 1992; Rosen and Beddington, 1993; Albertson et al., 1995; Morey, A.L., 1995; Swiger, and Tucker, 1996).
Hybridization histochemistry is generally amenable to combining with other techniques, such as immunohistochemistry, tract-tracing (Burgunder, and Young, 1988) and in vitro receptor autoradiography (Westlake et al., 1994). The combination with immunohistochemistry and/or tract-tracing may necessitate perfusion fixation of the animal (in order to preserve immunoreactivity and/or tracer deposition) prior to freezing the specimen and sectioning it. These sections, however, generally have a reduced signal-to-noise ratio for the hybridization histochemistry. The immunohistochemical steps are usually performed after the the hybridization histochemical ones to avoid loss of m

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