實驗概要
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualise the location of the antibodies.
Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, and may be used to analyse the distribution of proteins, glycans, and small biological and non-biological molecules. Immunofluoresence can be used in combination with other, non-antibody methods of fluorescent staining, for example, use of DAPI to label DNA. Several microscope designs can be used for analysis of immunofluorescence samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used. Various super-resolution microscope designs that are capable of much higher resolution can also be used.
主要試劑
1. Fresh 4% Formaldehyde
In 10mls ddH2O, add 0.8 g paraformaldehyde
2. Mounting Media
20mM Tris pH 8.0
0.5% N-propyl gallate
90% Glycerol
3. PHEM (500 mls) 2x
18.14 g Pipes
6.5 g Hepes
3.8 g EGTA
0.99 g MgSO4
pH 7.0 w/ KOH
4. PBS (5x in 500 mls)
20.45 g NaCl
0.465 g KCl
10.142 g Na2HPO4*7 H2O
0.545 g KH2PO4
pH 7.2
實驗步驟
1. Permeablize cells in 0.5 % TritonX-100 (freshly made, sonicated, and preheated to 37℃) in PHEM for 5 minutes alone
2. Slowly dribble in about 6 drops of fix (freshly made 4 % Formaldehyde/0.5 % Glutaraldehyde in PHEM) into Permeablization buffer. Let sit for another 2 minutes.
3. Fix in Fresh PHEM Fix for 20 minutes.
4. Rinse 3 times for 5 minutes each in PHEM.
5. Quench with Sodium Borohydride (a small pinch) in PHEM for 5 minutes. Be very careful not to let big bubbles form, which will damage fine actin structures, by lifting out every 10-20 seconds or so. The time will vary, so keep a close eye on it.
6. Quench 2 times in Sodium Borohydride (a small pinch) in PHEM (no Triton yet) for 4 minutes each following the same extreme caution as above.
7. Rinse 3 times for 5 minutes each in PHEM-T (PHEM 0.1 % TritonX-100 made fresh and heated to 37℃ (sometimes I sonicate and sometimes I forget, but it's probably better to be safe and sonicate to be sure the detergent is evenly distributed in the buffer).
8. ALL BLOCKS AND INCUBATIONS SHOULD BE DONE WITH THE COVERSLIPS FACING UP ON PARAFILM SO THE FINE STRUCTURES DO NOT GET DAMAGED.
9. Block at 37℃ in Boiled Donkey Serum for at least 45 minutes, ideally 60 minutes.
Note: you can leave in either block or in primary Antibody overnight at 4oC and it still looks beautiful.
10. Put on primary DM1A (mouse) at 1:300 in block (this concentration works great for my repeated frozen/thawed aliquot, but maybe if it's a fresh aliquot use it at 1:400) for 30 minutes at 37℃.
11. Rinse 3 times for 5 minutes each rinse in PHEM-T.
12. Put on secondary (I use Donkey anti-mouse-Rhodamine Red X) at 1:100 in block for 30 minutes at 37℃.
13. Rinse 3 times for 5 minutes each rinse in PHEM-T.
14. For Alexa 488-Phallodin, I use 3 uls/coverslip and I use about 150uls of block/coverslip to apply it. This timing is sort of crucial, only leave it in for 20 minutes at room temperature.
15. Rinse 1-2 times in PHEM-T
16. I do DAPI here... about 1 ul/10 mLs of PHEM-T for 60-120 seconds
17. Rinse 3 times in PHEM-T for 5 minutes each.
18. Rinse 1 time in PHEM for 5 minutes to get the bubbles off, etc (I don't really know why I do this step, but it feels right to remove the detergent for mounting).
19. Mount in 6-7 uls (I have a hard time avoiding bubbles so it helps to use this larger volume for me) of 90% Glycerol N-Propyl Gallate.