Immunohistochemistry has been used with both fluorescence and DAB stains for whole brain sections, as well as sections from organotypic brain slices. Rhodamine red goat anti-mouse as a fluorescent secondary antibody was used in paraffin embedded, organotypic slice culture and cryostat sections. DAB stain was used in
vivo and in vitro whereas organotypic slices were prepared for paraffin sectioning embedded for all target age groups.
7.2.7.1 Mouse on mouse (M.O.MTM) immuno-detection kit.
The Vector® M.O.M.™ immunodetection kit for mouse tissues has the advantage of very low background staining, which has been a problem in other studies (Chang 2009). Paraffin-embedded brain sections from mice aged 2-6 weeks were de-waxed through xylenes and rehydrated with different concentrations of ethanol 100 %, 95% and 70% respectively for 10 minutes each, then rinsed for 5 minutes in tap water. 10 mM sodium citrate buffer pH 6 was employed as antigen retrieval, which was necessary to uncover hidden antigen sites. For endogenous peroxide, slides were incubated in 3% H2O2 (hydrogen peroxide) for 5 minutes, then immersed in tap
water for 5 minutes. Blocking reagent was applied (block endogenous peroxidase activity) to block non-specific binding. Sections were washed three times for 5 minutes in PBS, and then in working solution of M O MTM diluent. The sections were incubated with primary antibody raised against the antigen of interest. Labelled secondary antibody was added, which is biotinylated anti-mouse M O M IgG Reagent, for 30 minutes. To perform Avidin/Biotin blocking, the avidin of biotinylated enzyme complex (ABC) is then added, which binds to the biotinylated secondary antibody (Figure 7-4). The incubation with a substrate for the enzyme generates a brown color resulting from DAB breakdown. Table 1 in the appendix summarizes the immunohistochemical process used for the paraffin embedded sections.
Chapter 7 Materials and methods
Figure 7-4 A diagram to illustrate the DAB reaction and how a labelled secondary antibody reacts with a primary antibody bound to a tissue antigen.
Immunohistochemical staining of paraffin sections (mouse on mouse kit – vector labs PK-2200). Incubation of brain tissue with primary antibody overnight at 40C is followed with a secondary antibody conjugated with a peroxidase enzyme for 1 hour. The ABC complex was added to tissue sections followed by DAB which is oxidized; the generation of brown color can detect under the light microscope. Longer incubations may result in an increase in background staining.
The diagram is adapted from php.med.unsw.edu.au/cellbiology/index.php?title=Group_3_Project- _Immunohistochemistry.
Chapter 7 Materials and methods
7.2.7.2 Immunohistochemistry technique for rhodamine-red fluorescent organotypic slice cultures
Organotypic slice cultures were fixed in 4% PFA for 20 minutes and then washed with PBS three times, for 5 minutes each. Slices were immersed in PBS and stored at 40C until required, which was be within two weeks of fixation. Slices were rewashed with PBS, and permeabilized by incubation with 0.5% Triton X-100 in PBS, in a humidity chamber at room temperature. Non-specific protein binding was blocked with 10% bovine serum albumin (in 0.5% Triton X-100 in PBS) for 2 hours at RT, and then washed with PBS. Slices were incubated overnight at 40C in primary antibody 1 µl:150 µl (anti-GFAP) in solution of 0.5% triton-X100 in PBS, then washed with PBS for 20 minutes. Rhodamine- conjugated secondary antibodies – (goat anti-mouse,1 µl:400 µl) in 0.5% triton-X100 in PBS was applied and incubated for 3 hours at room temperature. The tissue was then washed with PBS four times for 20 minutes, and finally incubated with DAPI stain for 20 minutes at room temperature. After washing in PBS 4 more times for 20 minutes, mounting medium was added to the slide and then cover slipped and sealed with nail varnish.
7.2.7.3 Immunohistochemistry technique for paraffin sections and organotypic slice cultures
Immunofluorescent and immunohistochemical staining were performed on both whole brain sections and organotypic brain slices. Organotypic slice cultures were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and immuno-fluorescent staining was conducted. Tissues were permeabilized with 0.5% Triton-X-100 in PBS for 1 h at room temperature. Nonspecific binding sites were blocked with 10% bovine serum albumin (BSA, Sigma) with 0.5% Triton-X-100 in PBS. Organotypic slices were incubated with anti-GFAP antibody (1:150 µl) in solution of 10% normal goat serum with 0.5% Triton-X-100 in PBS overnight (16-18 hrs) at 40C in a humidity chamber. Secondary antibody: Rhodamine red goat anti- mouse IgG (1:200 µl) in 10% normal goat serum with 0.5% Triton-X-100 in PBS was added to the slides and incubated for 3 hours in the humidity chamber at room temperature.
Chapter 7 Materials and methods
After washing with PBS, the slices were incubated with DAPI stain for 20 minutes at room temperature.
After dewaxing in xylene, the slides were immersed in three different concentrations of ethanol as follows: 100%, 95% and 70% and washed gently in running tap water. The sections were washed with PBS twice for 10 minutes in each container. The most important step is antigen retrieval, which involves steaming the sections for 40 minutes. This step was optimised several times to get the best result. The slides were left to cool for 20 minutes then blocked with 5% normal goat serum dissolved in 0.25% Triton X-100 in PBS for 2 hours in a humidity chamber at room temperature. The sections were then washed twice with PBS and incubated with primary antibody 1: 150 µl GFAP with 5% normal goat serum in PBS overnight, at 40C in a humidity chamber. The sections were again washed with PBS and incubated with rhodamine red- secondary antibody 1: 500 µl with 5% normal goat serum in PBS for I hour in the humidity chamber at room temperature. The sections were washed several times with PBS and then incubated with DAPI nuclear stain for 20 minutes. After washing with PBS 4- 5 times, slices were finally mounted in DABCO and coverslipped and sealed with nail varnish.