Rationale:
Immunostaining on formalin-fixed, paraffin embedded sections
has been revolutionized in 1991 by the discovery of heat-mediated retrieval
(Antigen Retrieval, AR) of immunoreactivity (Ref.
1,
2
). This method is now widely used and applies to the detection of the overwhelming
majority of antigens, with few exceptions for which enzymatic retrieval
is required (Ref.
3).
The method shown here uses a strong chelating agent, EDTA, (Ref.
4)
,and has been found efficient for the majority of antigens investigated.
More references are linked
here.
Immerse slides and holder in 1mM EDTA pH 7.5 (from a 100mM
stock) in a beaker. Cover with a piece of Saran wrap in which you made
holes. Put in a microwave oven and bring to a boil at max power (8 min
for 800 ml). Let boil for 15 min at a reduced power (Power 3) so that the
liquid continue to simmer. Cool at RT for 30 min to one hour. Transfer
to TBS.
Alternatively a pressure cooker or an autoclave can be
used, with optimal results.
You will perform all stainings in a humid chamber, as
the one depicted below.
NOTE: The moist chamber (see above) must fulfill the following criteria: a) be absolutely flat, b) maintain moisture over 3 days without drying. c) be washable.Double indirect AP immunohistochemistry (e.g rabbit anti antigen X, goat anti-rabbit AP, rabbit anti-goat AP)
1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).Top of Page
2- Briefly blot the slides without letting them dry and then apply 3% human or pig serum in PBS-BSA-NaN3 as a blocking agent (health hazard!).
3- incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking.
4- blot the slides without washing and apply the primary antibody (100µl), in a moist chamber, at RT for 1-18 hr.
5- Wash twice in TBS-T. 5 min / wash.
6- add the AP conjugated secondary antibody (50 to 100 µl) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use. SBA Goat anti mouse AP or Goat anti Rabbit AP can be used 1:200 in TBS-BSA- NaN3.
7- Wash thrice in TBS-T.
[8 and 9 are optional]
8- add the AP conjugated tertiary antibody (50 to 100 µl) and incubate for 15 min. The tertiary antibody should be absorbed against human serum; if not add 1% human serum before use. SBA Rabbit anti Goat-AP or Goat anti Rabbit AP can be used 1:300 and 1:200 respectively in TBS-BSA NaN3.
9- Wash thrice in TBS-T.
10- add 50 ml of the developing solution (see below ). Protect from direct light.
11- after 5 min, check the staining in your positive and negative controls.
12- check the staining at 10-15 min interval.
13- when staining is complete (usually < 1 hr), wash thoroughly in tap water.
14- preferably postfix in formalin for 4-5 hrs before mounting in water soluble mounting medium (glycerol gelatin).
AP Developing
solution:
For 50 ml developing solution add in order:
Indirect immunohistochemistry with avidin-biotin peroxidase(e.g rabbit anti antigen X, goat anti-rabbit biotin, avidin HRP)
1- Wash twice in TBS-T. 5 min / wash.Top of Page
2- Briefly blot the slides without letting them dry and then apply egg white in PBS-BSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) (Miller RT et al, Appl Immunohistochemistry 5(1): 63-66, 1997). Incubate for 10 min. minimum. (Use the yolk as directed in www.cucinait.com). Alternatively, use commercial blocking kits. Wash briefly once.
3- Apply 3% human or pig serum a s a blocking agent (health hazard!) for 10 min [after egg this is sometimes unnecessary]. Wash briefly.
4- Incubate with skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999). This is the free avidin blocking step. Wash twice in TBS-T.
5- blot the slides without washing and apply the primary antibody, in a moist chamber, at RT for 1-18 hr.
6- Wash twice in TBS-T. 5 min / wash.
7- add the biotin-conjugated secondary antibody (50 to 100 µl) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use. Most reagents are used at 1:100 or 1:200 dilution.
8- Wash twice in TBS-T.
8bis- block endogenous peroxidase by incubating in 0.1%NaN3 and 0.3% H2O2 for 30 min. Wash thrice.
9- add the HRP-conjugated avidin (50 to 100 µl, dilution 1:300 -500) (Dako P0364) and incubate for 20 min. Be careful not to dilute the avidin-HRP in biotin-containing or azide-containing medium.
10- Wash thrice in TBS-T.
11- add 50 ml of the developing solution (see below ). Protect from direct light.
12- after 5 min, check the staining in your positive and negative controls.
13- check the staining at 10-15 min interval.
14- when staining is complete (usually < 1 hr), wash thoroughly in tap water.
15- counterstain and mount in water soluble mounting medium (glycerol gelatin).
HRP Developing
solution:
For 50 ml developing solution add in order:
