What is the pre-treatment for a glutaraldeyde fixed iliac crest specimen?
What advantage do oligonucleotides have in in situ hybridization?
Are there rules for designing oligonucleotides?
How much do the results depend on the proteolytic pre-reatment?
Can I replace pepsin with proteinase K?
Are there alternatives for proteolytic pre-treatment?
Can I use ZytoFast probes on frozen sections?
Using my own probes, what are the crucial steps?
I have designed my own oligo probes. How can I determine their specificity?
How much care is necessary to avoid contamination with RNases?
Normally RNA degradation in paraffin embedded tissue occurs before fixation, e. g. due to long processing times before fixation of the specimen. In formalin fixed tissue, RNA is relatively stable. In a typical in situ hybridization experiment the steps before hybridization are sensitive to RNA degradation. However degradation requires a massive contamination with RNases. Baking of glassware, plugged tips, DEPC treatment of solutions, bench or other devices are not necessary. For a successful in situ hybridization experiment using paraffin embedded tissue the use of clean, possibly autoclaved material (pipette tips) and avoidance of coughing, sneezing and talking close to the specimen is sufficient.
What is the pre-treatment for a glutaraldeyde fixed iliac crest specimen?
If you need fixation with glutaraldehyde, the times for fixation and decalcification should be standardised. In our hands, using 24 h fixation in 2% glutaraldehyde followed by 3 d decalcification requires a proteolysis time of 50 min using 5 fold diluted ZytoFast pepsin for proper demasking of mRNA.
Which advantage do oligonucleotides have in in situ hybridization?
Oligonucleotides have a number of advantages in in situ hybridization. The most important are (a) precise control of melting temperature (b) avoidance of repetitive sequences, (c) easy and cheap synthesis (d) fast diffusion into the specimen (e) high stability of the probe and (f) - good reproducibility of labelling and hybridization reactions.
Are there rules for designing oligonucleotides?
ZytoVision uses its own, unpublished algorithm for the design of oligonucleotide probes for the use in in situ hybridization experiments. Generally oligonucleotides for use with ZytoFast systems should have: (a) a length of 28-32 nucleotides and (b) a GC content of 40-60%.
How much does my result depend on the proteolytic pre-treatment?
In situ hybridization is one of the most demanding methods in biotechnology and is influenced by a number of parameters. ZytoFast systems are of highest quality and precision and control most of the parameters influencing the result. However, as with all other in situ hybridization systems, ZytoFast systems are dependent on a proper proteolytic pre-treatment. This is crucial for the success of an in situ hybridization experiment. The determination of the optimal digestion time requires careful examination. For this purpose, ZytoVision offers special systems, which simplify evaluation of the proteolysis time necessary for a given specimen (Product number T-1007, T-1008).
Can I replace pepsin with proteinase K?
Yes. However, the proteolysis time necessary for proteinase K or any other proteinase must be evaluated for the specimen in question. Please note that the results received are not applicable for treatment with pepsin. Also, proteinase K has the disadvantage that enzyme activity is not easily standardised.
Are there alternatives for proteolytic pre-treatment?
Some publications describe boiling of the specimen in 10 mM citrate pH 6.0 using a microwave oven for unmasking RNA, as is done with various immunohistological stainings. Microwaving is not as easy to standardise and does not always give acceptable or transferable results.
Can I use ZytoFast probes on frozen sections?
In principle, the use of ZytoFast probes on frozen sections is possible. Quality control of ZytoVision does not test ZytoFast hybridization systems on frozen sections. In case you want to use frozen material you should postfix your section after proteolytic pre-treatment (5 min 4% PFA pH 7-8).
Using my own probes, what are the crucial steps?
You should use purified oligonucleotides labelled with terminal desoxynucleotidyl-transferase (Biotin, Fluorescein, Digoxigenin...). After labelling, oligonucleotides should be purified by precipitation. Be sure to have the correct hybridization buffer concentration after adding the oligos. ZytoVision offers labelling reactions using a modified labelling protocol for enhanced sensitivity and low background.
I have designed my own oligo probes. How can I determine their specificity?
There are different ways for controlling oligonucleotide specificity, which differ in terms of cost in time and meaningfulness. The simplest and best method is the use of the same material which is not expressing the target. Unfortunately this type of control does not often exist - Usually one or several of the following methods are applied: (a) DNase digestion using probes against DNA, (b) RNase digestion using probes against RNA, (c) oligonucleotides with several mismatches, (d), random oligonucleotides of the same nucleotide composition (e) sense oligonucleotides (RNA targets), (f) reverse oligonucleotides and (g) competition with unlabelled oligonucleotides.
