17.Protocol for immunostaining M.Aihara
this protocol is for neuron, astroglia and microglia but easily applicable for any samples
following procedures are done in room temperature
previous required materials and equipments (available from MA's stock)
cell samples on coverslip with flexiperm disc
PBS(-) (cold)
2% paraformaldehyde in PBS (cold)
Methanol (-20℃)
PBS(CaCl2, MgCl2,MnCl2 0.1mM respectively)+horse serum 10% (cold)
anti-fade(DABCO,sigma) (cold)
Fuji PROVIA ISO 1600
antibody
marker 1st 2nd wave length
neuron anti-MAP2(mono) 100× anti-mouse IgG-TRITC 50× 510-560nm
astroglia anti-GFAP(mono) 100× anti-mouse IgG-TRITC 50× 510-560nm
microglia isolectin B4-FITC 100× 450-490nm
(These antibodys and anti-rabbit IgG-FITC antibody are available from my stock! Please ask me.)
protocol
1. take blight-field phase-contrast photographs and note the location of imaging. The best way of fixing the image in your eyes is that you place the field at the center of distinctive type of cell and remember the cell.
2. mark the lattice of imaging with pen
3. wash 2 times with PBS(-)
4. fix cells with 2% paraformaldehyde in PBS(-) for 5 minutes
5. aspirate and dip into staining lack with -20C methanol for 30 seconds
6. add PBS(-), if you stop procedure in this step, you can preserve this plate in 4℃
7. blocking plate with 10% horse serum /PBS(Ca,Mn,Mg) for 20 minutes
8. leave in R/T light protected condition with 1st antibody in PBS/HS for 30 minutes
9. wash PBS(-) 3 times
10. add 2nd antibody with PBS/HS for 30 minutes in R/T light-protected condition
11. wash PBS(-) 3 times
12. add anti-fade in PBS
13. take photographs using Fuji PROVIA 1600 by adequate waves for light excitation
(if 1st antibody is labelled by any luminous molecule, omit step 10 and 11)
How to take fluorescent image with Nikon microscope TMD-300
14. switch on light of CAM230
15. set your desireble wave length of filter 1 (this dial is at the left side on lower panel of CAM230)
16. start up ARGUS/CA
17. change absorption filter to FITC or rodamin(TRITC) (this filter box is unter the objective lens of TMD300, change the lever from Ca to FITC or TRITC)
18. excitation filter open by controling on the monitor
19. you can see the fluorescent image (check the route of light "A", this port change dial is at the right side of TMD300)
20. take photographs after changing route from "A" to "B"
note: you had better to use Fuji PROVIA iso 1600 film.
If you can see easily fluorescent image, exposure time should be 2 or 3 minutes.
If you can see it with difficulty because of weak fluorescence, exposure time should be over 4 minutes.
Try and error!