2.5.2.1. Glial fibrillary acid protein
Immunostaining for GFAP is the prototypical method for labelling and imaging astrocytes (Sofroniew, 2009). Following transcardial perfusion, brains were sliced into serial frontal sections (40µm thick) which were immediately transferred to a freezing solution (sucrose 30%w/v, ethylene glycol 30%v/v in PBS) and stored at -80°C. Upon time of use, PLC sections were defrosted and rinsed 3 times for 10 min in PBS and incubated in blocking solution for 40 min at room temperature [10% normal goat serum (NGS) and 0.1% Triton X- 100 in PBS]. After this step, slices were rinsed once in PBS and incubated overnight either in negative solution [1% bovine albumin serum (BSA) and 0.1% Triton X-100 in PBS] or in primary antibody solution (rabbit polyclonal anti-GFAP 1:1000, DAKO) at 4°C. Following three 5-min washes in PBS, the slices were incubated with the secondary antibody [1:1000 alexa 488nm fluorescent secondary anti-rabbit (Invitrogen) and 1% BSA in PBS] for 2h at room temperature. The slices were washed 3 times in PBS for 5 min and mounted onto gelatine-coated slides and any excess liquid was removed. Vectashield mounting medium DAPI (Vector laboratories) was applied and coverslips were placed on the slides and sealed with nail varnish. The sections were stored at 2-4°C until imaged with the confocal microscope.
97 2.5.2.2. Calcium-binding protein B
Immunostaining for S100b is a method for labelling and imaging astrocytes (Sofroniew, 2009). Staining for S100b was performed on frozen slices from the PLC as previously described. Upon time of use, the sections were defrosted and rinsed for 10 min twice in PBS with a last wash in 5% triton diluted into PBS and incubated in blocking solution for 40 min at room temperature [20% normal goat serum (NGS) in PBS]. After this step, the slices were rinsed once in PBS and incubated overnight either in negative solution [1% bovine albumin serum (BSA) and 0.1% Triton X-100 in PBS] or in primary antibody solution [rabbit anti- S100b 1:1000, Abcam, in 1% BSA, 0.1% Triton X-100 in PBS] at 4°C. Following three 5- min washes in PBS, the slices were incubated with the secondary antibody [Alexa 488nm, 1:1000, anti-rabbit (Invitrogen), 1% BSA in PBS] for 2h at room temperature. The slices were washed 3 times in PBS for 5 min and mounted onto gelatine-coated slides and any excess liquid was removed. Vectashield mounting medium DAPI (Vector laboratories) was applied and coverslips were placed on the slides and sealed with nail varnish. The sections were stored at 2-4°C until imaged with the confocal microscope.
2.5.2.3. Neuronal nuclear antigen
NeuN is the prototypical method for labelling and imaging mature neurons (Gusel‘nikova and Korzhevskiy, 2015). PLC sections were sliced as previously described, defrosted and rinsed for 10 min 3 times in PBS and incubated in blocking solution for 40 min at room temperature [10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS]. After this step, the slices were rinsed once in PBS and incubated overnight either in negative solution [1% bovine albumin serum (BSA) and 0.1% Triton X-100 in PBS] or in primary antibody solution (mice monoclonal anti-NeuN 1:300, Millipore) at 4°C. Following three 5 min washes in PBS, the slices were incubated with the secondary antibody [1:500 alexa 488nm fluorescent secondary anti-mouse (Invitrogen)] for 2h at room temperature. The slices were washed 3 times in PBS for 5 min and mounted onto gelatine-coated slides and any excess liquid was removed. Vectashield mounting medium DAPI (Vector laboratories) was applied and coverslips were placed on the slides and sealed with nail varnish. The sections were stored at 2-4°C until imaged with the confocal microscope.
2.5.2.4. Co-staining of Doublecortin and GFAP
Immunostaining for DCX is the prototypical method for labelling and imaging immature neurons (Rao and Shetty, 2004) (Fig 2.13). In order to confirm that the DCX
98 immunoreactivity changes were associated with GFAP immunoreactivity impairments in the DG, we performed a co-staining for GFAP and DCX on frozen slices as previously described in the methods for GFAP staining. At time of use, the sections were defrosted and rinsed for 10 min twice in PBS with a last wash for 10 min in 5% triton diluted into PBS and incubated in blocking solution for 40 min at room temperature [20% normal goat serum (NGS) and 5% Triton in PBS]. After this step, the slices were incubated overnight either in negative solution [1% bovine albumin serum (BSA) and 0.1% Triton X-100 in PBS] or with the primary antibody solution (rabbit polyclonal anti-GFAP 1:1000, DAKO and mouse anti DCX, 1:250, Santa-cruz and 1% BSA, 0.1% Triton X-100 in PBS) at 4°C. Following three 5 min washes in PBS, the slices were incubated with the secondary antibody [Alexa 488nm, 1:1000, anti- rabbit (Invitrogen), Alexa 546, 1:1000, Life technologies and 1% BSA in PBS] for 2h at room temperature. The slices were washed 3 times in PBS for 5 min and mounted onto gelatine-coated slides and any excess liquid was removed. Vectashield mounting medium DAPI (Vector laboratories) was applied and coverslips were placed on the slides and sealed with nail varnish. The sections were stored at 2-4°C until imaged with the confocal microscope.
Figure 2.13: Neurogenesis
First, progenitor cells proliferate for 1-3 days leading to either apoptosis or to neuronal differentiation into immature neurons (4-7 days). Immature neurons express DCX and they mature into neurons expressing NeuN (4-6 weeks).
99 2.5.2.5. Ionized calcium binding adaptor molecule 1
Iba1 is a 17-kDa EF-hand protein expressed specifically in microglia and macrophage (Imai et al., 1996). Iba1 has actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia (Ohsawa et al., 2004). Immunostaining for Iba1 was used to quantify the microglia number and performed on frozen slices from the PLC obtained as previously described. Upon time of use, sections were defrosted and rinsed 3 times for 10 min in PBS and quenched for 20 min in a hydrogen peroxide solution (1% H202 and 5% methanol in PBS). After three 5-min washes with PBS, the sections were incubated in blocking solution for 45 min at room temperature [10% normal goat serum (NGS) and 0.3% Triton X-100 in PBS. After this step, the slices were incubated overnight either in negative solution (5% NGS and 0.05% Triton X-100 in PBS) or with the primary antibody solution (rabbit polyclonal anti-IBA1 1:3000, Vectastain, Elite vector ABC kit; Vector laboratories, 0.05% triton X-100 PBS and 2% of NGS into PBS) at room temperature. On day 2, Following three 5-min washes in PBS, the slices were incubated with the secondary antibody [with 4 drops of biotinylated goat anti rabbit (Vectastain, Elite ABC kit; Vector laboratories, 6 drops of NGS into 40ml of PBS] for 90 minutes in the dark at room temperature. The slices were washed 3 times in PBS for 5 min and incubated for 90 minutes with avidin-biotin complex [3 drops of A and 3 drops of B (Vectastain, Elite ABC kit, Vector laboratories) into 30ml of PBS prepared 30 min in advance]. Following three additional 5-min washes with PBS, the slices were incubated for around 2 min with 3,3'-Diaminobenzidine (DAB solution; 4mg/ml, 2ml of DAB solution for 40ml of PBS with 12μl of H202). To stop the DAB reaction, the slices were washed for 5 min into distilled water followed by two 5-min washes into PBS. Lastly, the slices were mounted onto gelatine-coated slides and allowed to dry overnight after any excess liquid was removed.
On the third day, the slides were washed into distilled water and dehydrated in increasing concentrations of ethanol (70%, 90%, and 100%) for 2 min and finally Xylene for 1 min. DPX mounting media was then placed on the slides and a coverslip carefully placed on top. The slides were then allowed to dry for 24 h before being imaged.
2.6. Image analysis