Double staining of microtome serial section

DOUBLE STAINING OF MICROTOME SERIAL SECTION

1. Double staining of microtome serial section

2. Processing of serial sections for double staining Sections in ribbon on slide Dewax in xylene Xylene Ab.alcohol mixture Ab.alcohol Water

3. Stain Wash Dehydrate Counterstain Differentiate Xylene Mount in canada balsam

4. Methods 1. Affix ribbons on slides Clean slide properly Rinse them with 95% alcohol Dry then by evaporation Place a tiny drop of adhesive on the dry slide &spread it over by fore finger-place the slide flat on the table Flood the slide with distilled water Cut ribbons into convenient lengths Place ribbons on the slide with the glistening slide down

5. Pass slide over a flame /warm them with gentle sliding underneath until the sections flatten out without melting Drained off water &mark the slide Left for dryining & then store properly 2 Staining Remove paraffin from sections( dewaxing ) using xylene /benzene for this , the slide is dropped into a coplin jar containing dewaxing agents Take care to ensure which slide carries the sections& avoid rubbing of them

6. After about a dozen slide have been dewaxed , replace with fresh fluids Transfer to absolute alcohol , xylene mixture Transfer to absolut alcohol &then to water If smoky precipitates are appeared , replace the alcohol Sure test to make sure whether all wax has been completely removed If water flows evenly over the surface including the sections ,they are free from wax Wash thoroughly with water Stain with suitable dye

7. Pass through required dehydrants Counter stain Transfer to xylene Mount in balsam &dry by evaporation method, on the surface of a hot plate. Balsam act as a varnish &prevents the slides from getting dried out Place cover slip on the slide surface on the next day Before placing, the coverslip should be passed over a flame

8. 3 Cleaning When the slides are completely dried , the surface balsam should be carefully removed with a blunt knife &left over night If the freshly cut edge appears to be sticky, the slide is not yet ready If not ,the whole slide is dipped in 90%alcohol &rubbed briskly to remove excess balsam. If is immediately rinsed in a strong soap solution & the polished

9. 4.Labelling Write serial no . of the slide Attach the label written in water proof India ink Adhesive Fixatives used for fixing the paraffin ribbon on the slide Standardised adhesives are Mayer’s adhesives and Haupt’s adhesives Commonly used adhesives are gum- arabic , commercial albumen or albusol , fresh egg white, sheet gelatin etc

10. Should apply using a toothpick/glass rod Mayer’s adhesive Drain the white of an egg into a graduated cylinder Add equal volume of water +same volume of glycerine Add 0.5% sodium benzoate/1% sodium salicilate Shake well& filter

11. Haupt’s adhesive Dissolve 1g sheet gelatin in 100ml water Add 0.5g sodium benzoate or 2g phenol Store properly Double staining saffranin -fast green method Material:- strobili of lycopodium / selaginella Fix in chromoacetic,medium-24hrs Wash in running water (24 hrs)

12. Dehydration :- 30%alcohol(5min),50%alcohol (2min),70%,90%,100% alcohol (1min each) Transfer through xylene series Infiltrate with paraffin of M.P 53-55 ‘c Embedded in paraffin Cut sections of (10-12 u m) Affix on the slide selecting only median sections Dewax in xylene Transfer to xylene - ab. Alcohol (1:1) Transfer to ab.alcohol ,then to water Stain in safranin (5min)

13. Wash in water Dehydrate through 59,70,80,90,100% of alcohol (2 min each) Counter stain with fast green in clove oil(1/2 min) Wash in clove oil Clear in xylene (quick) Mount in canada balsam /DPX In this method, down series is used for staining while up-series is used for counter staining &mounting

14. Down series(staining) Dewaxing Xylol-ab alcohol 3:1 Xylol alcohol 1:1 Xylol alcohol 1:3 Ab. Alcohol Alcohol.75% Alcohol.60% Alcohol .40% Alcohol .30% Distiled water Staining( safranin ) Wash in water

15. Up series (counter staining &mounting) Alcohol.30% Alcohol .45% Alcohol.60% Alcohol,75% Alcohol .95% Ab. Alcohol Counter staining(fast green in clove oil) Clove oil Xylol wash Mounting

16. Thank you