V. Trinkaus-Randall
Histological Techniques
To fix cells for in situ hybridization
(adapted from Singer et al Biotechniques 4(3) 230-250 1986
*rinse cells gently in PBS (1x).
*Fix in freshly prepared 4% paraformaldehyde in PBS/ 5mM MgCl2 pH7.4 for 15 min rm temp
(After adding MgCl2 filter)
*wash cells with PBS/5mM MgCl2 pH 7.4 3x. Cells can be stored for 2 weeks or put in
70% and store at 4oC.
Prepare cells
*Rehydrate cells in PBS/5mM MgCl2 for 10 min on belly dancer
*Start control at this point and leave the others in the above buffer!!!!
for control using RNAse
1. Incubate cells in RNAse (100 ug/ml) for 1 h at rm temp
Dilute 20x RNAse stock with 2x SSC (Sigma 2 mg/ml). After 1 h keep these slides
marked and follow through with the procedure.
*Replace solution on experimental cells and on cells without probe with 0.2M Tris-HCl pH
7.4/0.1M glycine for 10 min on rocker. Meanwhile heat 2x SSC at 65oC
* Replace solution with 50% deionized formamide in 2x SSC for 15 min on a heat block.
Meanwhile heat 100% formamide at 90oC and prepare probe mix
Take lyophilized probe (ie. 100 ng) and dissolve in 320 ul 100% formamide 90oC.
Immediately before hybridization add an equivalent volume (320 ul) of the hybridization volume
containing 20x SSC, 2% BSA, 50% dextrand sulfate and VRC at a ratio of 1:1:2:1:
Place hybridization mix on cells (make sure cells covered for a 8 well slide will need about 150
ul/division)
Note for probe controls do not add probe!!!!
Parafilm the chamber slides and put in a humidified chamber at 37oC for overnight.
Washing
*50% formamide/2x SSC for 30 min at 37oC
*50% formamide/1xSSC for 30 min at 37oC
*1xSSC at rm temp on belly dancer and can be left in this solution overnight
Detection with anti-digoxigenin (rhodamine or fluorescein) Fab fragment
*Wash cells in buffer for 10 min on belly dancer
buffer = 1x PBS containing 0.5% BSA pH 7.4
*Incubate the cells with 100 ul of anti-dig (1:4) in a humidified chamber at 37oC for 1 h
*Rinse cells in buffer 5x
*wash cells 3x for 10 min in buffer
*check under microscope and if there is background keep washing
*mount in anti-fade media, wrap with foil and keep in fridge
to fix cells for immunohistochemical staining of proteins
culture on glass coated slides or plastic that does not autofluoresce
wash with PBS
fix in 4% paraformaldehyde (freshly made) or 3.7% formaldehyde - diluted in PBS for 15 min at
rm temp (freshly made again) (other fixation protocols may need to be employed…ie. methanol,
aetone, glutaraldehyde…)
Fix for 1 hr if it is tissue and not cell cultures
wash in PBS once
permeabilize with (0.1% Triton -x for 3 min) (If the protein that you are looking for is on the
membrane you do not need to permeabilize)
wash with PBS and if stopping here store in PBS containing 0.1% sodium azide, parafilm and
keep at 4oC
Staining cells
keep solutions on ice
need:
PBS
3% milk solution (filtered) and made fresh (note BSA can also be used - check in product info –
if using biotinylated avidin use BSA – in all cases it should be fresh and filtered)
1% milk solution "
"
primary antibody diluted in 1% milk solution
wash cells carefully
incubate cells in 3% milk solution for 5 min
remove and add primary antibody and incubate overnight on rocker at 4oC
wash in PBS several times (5-7x)
incubate in PBS for 10 min
incubate in 3% milk solution for 5 min
incubate in secondary antibody diluted in 1% milk for 1 h on rocker at room temp
wash 5x with PBS
incubate with PBS for 5 min 2x
mount with fluorescent quenching medium
to stain with propidium iodide for nuclear staining (for DNA only use RNAse)
take stock solution ( 5 mg/ml) and dilute to 0.5 ug/ml and stain for 2-5 min at rm temp
wash and cover slip
To fix cells for TEM
Use Karnovsky’s buffer
Do not switch buffer systems (ie Tris to PBS)
Controls for Immunofluorescence are extremely important as they are for westerns!!!!!!
When you use the confocal – it sees low level fluorescence – controls must be negative
!!!!!!!!