Material and Methods

4.1 Animals

Animals were handled according to procedures approved by the Comité de Protection des Animaux du CHU under the guidelines of the Canadian Council on Animal Care. Mice were maintained in a temperature-controlled room (~23°C) with a light/dark cycle of 12/12h. Experiments were performed during the light period and all animals had access to food and water ad libitum. Four groups of were used: WT, P301S, TKO and hTau mice.

WT mice (C57BL/10J, #000665, Jackson Laboratory, Bar Harbor, ME, USA) are used as non-transgenic controls to observe tau phosphorylation. P301S mice (B6;C3-Tg(prnp-MAPT*P301S)PS19Vle/J, #008169, Jackson Laboratory, Bar Harbor, ME, USA) were used as transgenic mice model, the latter showing widespread neurofibrillary tangles and tau pathology. TKO mice (Mapttm1(EGFP)Klt/J, #004779, Jackson Laboratory, Bar Harbor, ME, USA) were used as negative controls for phosphorylation and aggregation due to the absence of tau protein. These mice have a knock-in in the first exon of MAPT gene, disrupting its expression. hTau mice (B6.Cg-Mapttm1(EGFP)KtlTg(MAPT)8cPdav/J, #005491, Jackson Laboratory, Bar Harbor, ME, USA), result of mating of 8c mice and tau knock-out mice, are designed to only express human tau isoforms on a murine tau knock-out background. This line was used in this study to compare the phosphorylation of mice tau with human tau, depending the fixation method. Half of the mice were hypothermic and were used as a positive control for tau hyperphosphorylation. Hypothermic mice were put in a chamber with isoflurane for anesthesia to induce hypothermia-mediated tau hyperphosphorylation.

4.2 Fixation

Hypothermic mice were killed by decapitation when their body temperature was low enough (under 30°C) after anesthesia or transcardially perfused with a saline solution 0.9%

for this condition. Non-hypothermic mice were killed by decapitation without anesthesia to avoid hyperphosphorylation. For the comparison between Bouin and PFA, both hypothermic and non-hypothermic mice (WT, TKO and hTau) that were killed by

decapitation without perfusion had their brain immediately removed and cut in half, one hemisphere being drop-fixed in Bouin and the other in PFA 4%. When the effect of perfusion was studied, mice were briefly put in isoflurane chamber until completely anesthetised. Then, their body temperature was monitored using a rectal probe (Thermalert TH-5, Physitemp, Clifton, NJ, USA) to make sure they are not hypothermic and rapidly perfused with a saline solution 0.9%. Then, the brain was removed and each half-hemisphere were drop-fixed in each fixative. Brains were cut in half on ice, and half of each fixatives was kept ice-cold (4°C) and the other half at room temperature. Brains were kept in fixative on a rotarod for 24h, followed by three washes of PBS 0.1M pH 7.4. Brains were embedded in paraffin blocks and 10 µm thick sagittal sections were processed for immunostaining analyses.

4.3 Immunohistochemistry

Prior to immunostaining, slides were warmed at 60°C for 10 minutes and deparaffinised with Citrisolv (22-143-975, Fisherbrand) for 2 x 10 minutes. Rehydration of sections was made by immersion in graded ethanol solutions (100%, 95%, 70% and 50%) for 5 minutes each. After rinses in distilled water, antigen retrieval was performed by incubating slides in citrate buffer pH 6.2 for 30 minutes at 80°C, then letting slides cool down for about 1h at room temperature. Three washes of 8 minutes in PBS 0.2M pH 7.4 followed, then the slides were quenched with endogenous peroxidases using a solution of PBS containing 3% of peroxide for 30 minutes, and washed again in PBS for 3 x 8 minutes. Non-specific background staining was blocked for 1h in a solution of PBS consisting of 4% normal goat serum, 4% Triton X-100 10% and 1% bovine serum albumin. Sections were incubated overnight at 4°C in primary antibodies (mouse anti-CP13 (phospho S202), 1:200 dilution [Peter Davies]; mouse anti-AT8 (phospho S202/T205), 1:200 dilution [MN1020, Thermo Fisher Scientific]; mouse anti-PHF1 (phospho S396/404), 1:20 dilution [Peter Davies];

mouse anti-MC1, 1:25 dilution [Peter Davies]; rabbit anti-TauC (total Tau), 1:1000 dilution [A0024, Dako]) diluted in a solution of PBS with 1% normal goat serum and 0.4% Triton X-100 10%. The sections were afterwards washed in PBS 3 x 8 minutes and incubated with the appropriate biotinylated secondary antibody (biotinylated goat anti-mouse, 1:1500

dilution [BA-9200, Vector Laboratories]; biotinylated goat anti-rabbit, 1:1500 dilution [BA-1000, Vector Laboratories]). Tissues were washed again in PBS 3 x 8 minutes and incubated with avidin-biotin complex (Vectastain ABC kit, PK-6100, Vector Laboratories) for 1h. Washes with PBS 3 x 8 minutes followed, and sections were washed with Tris-HCl 0.05M pH 7.6 for 5 minutes. Visualisation was made with 3-3’ diaminobenzidine tetrahydrochloride, also known as DAB (D5905, Sigma-Aldrich) in 0.05% hydrogen peroxide and Tris-HCl. Sections were washed again in PBS 3 x 8 minutes, rehydrated in graded ethanol solutions (50%, 70%, 95%, 100%), then Citrisolv and finally mounted with Neo-Mount anhydrous mounting medium (1.09016.0500, Millipore). Immunolabeled tissues were observed under a Carl Zeiss AxioCamIC (Carl Zeiss, Jena, Germany) microscope.

4.4 Immunofluorescence

Paraffin-embedded sections of mice brains were warmed at 60°C for 10 minutes and deparaffinised with Citrisolv for 2 x 10 minutes. Rehydration of sections was made by immersion in graded ethanol solutions (100%, 95%, 70% and 50%) for 5 minutes each.

After rinses in distilled water, antigen retrieval was performed by incubating slides in citrate buffer pH 6.2 for 30 minutes at 80°C, then letting slides cool down for about 1h at room temperature. Three washes of 8 minutes in PBS 0.2M pH 7.4 followed, then non-specific background staining was blocked for 1h in a solution of PBS consisting of 4%

normal goat serum, 4% Triton X-100 10% and 1% bovine serum albumin. Sections were incubated overnight at 4°C in primary antibodies (mouse anti-CP13 (phospho S202), 1:200 dilution [Peter Davies]; mouse anti-AT8 (phospho S202/T205), 1:200 dilution [MN1020, Thermo Fisher Scientific]; mouse anti-PHF1 (phospho S396/404), 1:20 dilution [Peter Davies]; mouse anti-MC1, 1:25 dilution [Peter Davies]; rabbit anti-TauC (total Tau), 1:1000 dilution [A0024, Dako]) diluted in a solution of PBS with 1% normal goat serum and 0.4% Triton X-100 10%. The sections were next washed in PBS 3 x 8 minutes and incubated with the appropriate fluorophore-conjugated secondary antibody (Alexa Fluor 488 goat mouse IgG, 1:1500 [A11029, Life Technologies]; Alexa Fluor 568 goat anti-mouse IgG, 1:1500 [A11031, Life Technologies|; Alexa Fluor 488 goat anti-rabbit IgG,

1:1500 [A11034, Invitrogen]; Alexa Fluor 568 goat anti-rabbit IgG, 1:1500 [A11036, Invitrogen]). Tissues were washed again in PBS 3 x 8 minutes and incubated in a solution of PBS containing DAPI (D3571, Life Technologies) for 15 minutes. Sections were washed in PBS for 8 minutes, then in ethanol 50% for 5 minutes and covered with Autofluorescence Eliminator Reagent (2160, EMD Millipore) protected from light for 5 minutes. Slides were washed with ethanol 70% for 3 x 1 minute and mounted with Fluoromount-G (0100-01, Southern Biotech). Immunolabeled tissues were observed under a Carl Zeiss AxioCamIC microscope.