Ab134014

Immunohistochemistry for the visualization of neuronal nuclear antigen (NeuN), rat endothelial cells antigen 1 (RECA-1), and Iba-1 positive cells expression was performed. To begin the procedure, six sections on average were selected in each brain region, after being blocked with 10% normal goat and rabbit serum for 1 h. In brief, the sections were incubated overnight at 4 °C with a NeuN antibody (1:1000, ab134014, Abcam, Cambridge, UK), RECA-1 antibody (1:1000, ab9774, Abcam, Cambridge, UK), Iba-1 antibody (1:500; ab5079, Abcam, Abcam, Cambridge, UK), and 4HNE (1:200; HNE11-S, Alpha Diagnostic International, San Antonio, TX, USA). The sections were then incubated with the biotinylated rat and mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) with 0.3% Triton X-100 in PBS for 2 h at RT and subsequently incubated with antibody–biotin–avidin–peroxidase complex solution (Vector Elite ABC kit^®; Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Finally, the sections were stained with a 3.3′-diaminobenzidine tetrahydrochloride (DAB kit; Vector Laboratories, Burlingame, CA, USA). The sections were finally mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount^® (Vector Laboratories, Burlingame, CA, USA).

Stereotactic injections of brain extract were performed as described previously with minor modifications37 (link). WT mice (male, 3-months-old, male, C57BL6/J) were injected by using a 30-gauge Hamilton microsyringe in the left frontal cortex (bregma +1.3 mm, 1.5 mm lateral to midline, −1.6 mm relative to bregma) at an infusion rate of 0.2 μl min⁻¹. HMW (2.5 μl) (Frc. 2–3) or LMW (Frc. 13–14) SEC fractions (100 or 500 ng ml⁻¹ human tau) from rTg4510 brain extract (male, 12-months-old, PBS-soluble, 3,000g) were injected. The same volume of PBS was injected as a negative control. Mice were killed 48 h after injection and brain sections from frontal cortex were immunostained with human tau-specific antibody (Tau13, #MMS-520R, Covance, 1:2,000), chicken polyclonal anti-NeuN antibody (#ab134014, Abcam, 1:500), and counterstained with DAPI. Anti-mouse Alexa488 (1:1,000) and CY3-labelled anti-chicken IgG (1:1,000) secondary antibodies were used to detect human tau and NeuN, respectively (see also Immunostaining of brain sections). Images were acquired on an AxioImager Z1 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany). Images were semi-quantitatively evaluated for human tau staining by a rater who was blinded to the experimental conditions, using a score from 0 (no human tau labelling on NeuN positive neurons) to 4 (maximum human tau labelling on NeuN positive neurons).

Goat anti-IGF-1 antibody (ab106836), rabbit anti-ps198 antibody (ab79540), chicken anti-NeuN antibody (ab134014), and rabbit anti-GFAP (ab33922) were purchased from Abcam (Cambridge, MA, United States). Rabbit Polyclonal GSK3 Beta Antibody (22104-1-AP) was purchased from Proteintech (Cambridge, CA, United Kingdom). Rabbit Anti-phospho-GSK3 Beta (Tyr216) antibody (bs-4079R) was provided by Bioss (Danvers, MA, United States). Donkey anti goat HRP and goat anti rabbit HRP antibody (PAB0012 and PAB0011) were purchased from Bioswamp (Waltham, MA, United States). For secondary antibodies used in immunofluorescence, we used the Alexa Fluor 488 and 594 from Abcam (Cambridge, MA, United States).