We have used the following steps for IHC but the background is major issue in analysing the data. Image were taken by LSM 780 and analysed by Image J software.
The steps
1. 3 Mm punch biopsy tissue : 4-8 hr formalin fixed.
2. Dehydration
3. Paraffin wax embedded
4. Section(5µm) cut on Poly L lysin coated slide.
5. water in between the slide and tissue section
6. Heat dried 75°C 30 min,
7. Xylene-1 incubation 10 minutes
8. Xylene-2 incubation 10 minutes
9. Alcohol 100 % incubation 5 minutes
10. Alcohol 95 % incubation 5 minutes
11. Alcohol 85 % incubation 5 minutes
12. Alcohol 70 % incubation 5 minutes
13. Transfer in 200 ml distilled water
14. Transfer in 100 ml 1X sodium citrate buffer incubation 60°C, over night
15. Place for cooling in RT
16. Mark with hydrophobic pen
17. Wash with PBST 3 times 5 minutes each
18. Add protein Blocker (DAKO SERUM FREE PROTEIN BLOCK X 0909) for 5 minutes
19. Add Primary antibody 100µl/ well incubation-4°C over night, dark
20. Carefully Wash with PBST 3 times 5 minutes each, the Antibody should not be intermixed
21. Add Secondary Antibody in all the sections, incubation-4°C 30 minutes, dark
22. Wash with PBST 3 times 5 minutes each
23. Add 2 drop of DAPI (Vectashield H-1200) on slide, gentle place the bigger cover slip so that each section can be covered and place gentle in the staining box, undisturbed
24. Cover two edges with nail paint, let it should be dry
25. cover remaining sides with nail paint
26. Let it should be dried and ready for use under microscopy.
In the uploaded picture No primary antibody have been used even though the secondary antibody is binded..
The numbers associated with my suggestions below correspond to that of yours in the left margin.
1. A minimum of 6 hours is required to obtain an acceptable quality of fixation in 10% buffered formalin.
2. Are you using tissue processor equipment to process the post-fix tissue specimens.
4. Instead of using Poly-L-Lysin coated slides, I highly recommend the use of the 'supperfrost plus' slides from Fisher or VWR.
6. Incubation of slides at 75 degree Celsius in a 'dry atmosphere' may cause introduction of tissue artifacts. Please dry the slides at ambient temperature for about 10 hours ( preferably 24 hours ). Please bake the slides at no higher than 59 degree Celsius 2 to 3 hours.
14. Incubation of slides in sodium citrate buffer, pH 6.0, at 60 degree Celsius may be insufficient to unmasking of the target epitope(s). I recommend the use of a pressure cocker/microwave oven ( J. Histochem. Cytochem., 43: 193-201, 1995).
19. I would recommend the application of the primary antibody at different dilutions (concentrations) to obtain an optimum specific staining without or minimum non-specific background staining.
23. Not clear to me.
Depending on how high your laser power and PMT gain were set, this may just be autofluorescence. If you hit biological material with enough light, you will get signal. If you use the same settings that were used for your positive control, what do you see?
To reduce autofluorescence try a shorter fixation time. Also, before the incubation with antibodies, incubate the sections in 50 mM ammonium chloride for 30 min (Neuroscience 274:209-17, 2014); this reduces background fluorescence.
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