Immunostaining

(4 wells chamber slides)

 

Before fixation, wash the cells with PBS

 

I-                  Fixation (3 possibilities)

 

Volume : 300 to 400 ml

 

1-     Paraformaldehyde

Fixation with PFD 2 %: dilute PFD 4% with PBS/glycine, or with CSK buffer (if extraction?).

            -30-40 min at RT with 2% PFD (or 15-20 min RT with PFD 4%)

            - Possible O/N (or a couple of days) at 4C, but the staining should be less good.

Reversible.

 

2-     Methanol/acetone (1:1 v) (-20)

Most commonly used, M/A will extract lipids and soluble proteins, cannot wait!

-         10 min. at –20C or RT (but the M/A must be stored at –20)

 

3-     Triton extraction + paraformaldehyde

 

*For nuclear immunostaining, for proteins of the cell skeleton

 don’t forget to use protease inhibitors

 

A/ triton extraction : 0.5 % triton in CSK buffer + protease inhibitors

-         20 min for 3d, 15 min for On top, 10 min for 2d

-         Wash quickly with CSK buffer+protease inhibitors without triton (2X 5 min)

-          

*Triton: peroxide-free, Boehringer Mannheim #1332481, 10% stock at –20

  can be frozen and thawed many times

  air sensitive.

 

CSK buffer  : NaCl 100 mM, sucrose 300, mM Pipes 10 mM pH 6.8, MgCl2 5 mM

+ NaF 0.25mM final (250mM stock)

+ aprotinin 0.01mg/ml final (10mg/ml stock)

+ PMSF 1mM (100mM stock) or pefablock (?)

 

            B/ Paraformaldehyde fixation

-         4 or 2% in CSK buffer, warm up at RT (15 min) before use

-         20 min at RT

 

II-               Wash

 

Remove the fixation buffer

3X 20 min with PBS/Glycine (50mM, RT, CHECK)

 

III-            Blocking

 

-         10% Goat serum (GS) in IF buffer 1 hour at RT or O/N at 4 degree

 

*When matrigel, you can: Block immunoreactive mouse Ig species in EHS for an additional 30 minutes at RT with anti mouse Fab, #H152 in Bissell Human book (From Caltag # M35000, Goat anti-mouse IgG Fab fragments), stored in –20 in human room, use 1/100.

The #152 Fab can be kept in 4 degree during 3 month.

 

*If background in the cells (stained nuclei), you can also use at this step an anti-human Fab fragment (Jackson lab) (#97, 4 degree in human room), use 1/100.

 

 

IV-            Primary antibody

 

Ideal volume: 300 ml

 

*You can reduced the volume up to 100 ml, for 2d, but then you should gently shake (or rotate gently) your sample to be sure that the entire surface of each slide will be in contact with the primary antibody solution.

 

*When you are using a primary antibody for the first time, you should do a titration around the recommended dilution for immunostaining or around 1/10 of recommended dilution for Western Blot.

 

-         IF buffer + 10% GS       + primary antibody at the right dilution

+ anti mouse Fab #152 1/100 if matrigel

+/- Anti human Fab #97 1/100

-         With matrigel, incubated 2 hours at RT (1 hour for 2d) or O/N at 4 degree celsius.

 

*If blocking has been done O/N, try to avoid doing your primary O/N and vice versa. It could be better to do the primary O/N (longer time with the antibody and reduce background, but matrigel can become liquid depending on the lot used!)

 

IF buffer composition :

 

V-               Wash

 

Remove the primary antibody solution

3X 20 min with IF buffer, gentle agitation

 

VI-            Secondary antibody (in the DARK)

 

-         IF buffer + 10% GS       + secondary antibody at the right dilution

 

-         Incubate 40 min. to 1 hour max (otherwise it it induces high background), covered from light.

 

VII-         Wash

 

Remove the secondary antibody solution

3X 20 min with IF buffer, gentle agitation

 

VIII-      Nuclear staining

 

Nucleus                2nd Ab

FITC

Texas Red

Cyanine (IR)

DAPI

x

x

x

Propidium iodide (confocal)

x

 

x

Picogreen (confocal)

 

x

x

Topro 3 (confocal)

x

x

 

 

 

1-     DAPI (UV)

 

Dilute the stock solution 1:10000, i.e. 1 ml in 10 ml of PBS (DAPI stock solution : 5 mg/ml), store the working solution for up to 1 month at 4 degree.

Add 300 ml of DAPI working solution, 5 to 10 min, RT, dark.

 

2-     Propidium iodide (red, use texas red filter)

 

* in that case the secondary antibody should be FITC.

Dilute the stock solution to have 1 mg/ml, i.e. 1:50 (stock solution : 50 mg/ml, CHECK)

Add 300 ml of propidium iodide working solution, 20 min, RT, dark

 

3-     Hoechst (UV, living cells)

 

4-     Picogreen (green, use FITC filter)

 

* in that case the secondary antibody should be texas red.

Dilute the stock solution 1:4000.

Add 300 ml of picogreen working solution, 20 min, RT, dark

 

5-     Topro 3 (IR)

 

Dilute the stock solution 1:5000.

Add 300 ml of Topro 3 working solution, 20 min, RT, dark

 

IX-            Wash

 

Remove the nuclear staining buffer.

2X 10 min with IF buffer, RT (not necessary for DAPI, just add IF buffer once)

 

X-               Mounting medium

 

Remove the upper part of the chamber slide, prepare the mounting media, use coverslip #1

 

1-     Vectashield

 

One drop on the sample, then put the coverslip on the slide. You may need to press lightly on the coverslip with paper to remove the excess of vectashield.

Then add the nail polish to seal.

Let it dry (5-10 min.)

 

2-     AntiFade (molecular probe)

 

Stored at the –20 (human room).

You will have to reconstitute the antifade a few minutes before using it :

-         put the buffer at 37 degree,

-         fill the antifade tube up to 1 ml and use the 1ml pipet to resuspend

-         wait for the buble to disappear

-         Add two drops on each sample

-         Put the coverslip #1

-         Dry by pressing with paper, you don’t need nail polish.

-         Let it dried at RT for 24 hours (no more than 36 hours) for the 3d or O/N for the 2d, in the dark.

-         Once it’s dryed : store at – 20 degree (non cycling freezer)