Thioflavin s thios

Sections of all above-described animals were fixed and stained as described above, excluding the endogenous peroxidase blocking step, ABC step and DAB visualisation. Primary antibodies are listed in Table 1. Secondary antibodies used were donkey anti-goat and donkey anti-rabbit, both coupled to either Alexa 488 or Alexa 594, (dilution 1:400, Invitrogen, Camarillo, CA, USA). For the detection of β-pleated sheets, sections were incubated with 1% Thioflavin S (ThioS, Sigma, St. Louis, Missouri USA) in milliQ for 5 minutes, washed three times with 70% ethanol and two times with TBS. Sections were mounted with Vectashield® (Vector laboratories Inc) or PVA-DABCO^® mounting medium (Sigma). Between incubation steps, sections were washed extensively with TBS. A Leica TCS SP2 AOBS confocal laser scanning microscope (Leica Microsystems) was used to visualise the immunofluorescence. To exclude bleed-through of fluorescence emission, a series of images was obtained by sequential scanning of channels through a 40× lens (zoom factor 1× or 2×, resolution 1024 × 1024). Staining was performed and analyzed on at least three sections per animal. In the figures, a representative image of these stainings is shown.

Brain sections (40 μm) were cut using a vibratome and stained using modifications of standard procedures previously described [33 (link)]. Staining was performed in a limited number of batches that were balanced across experimental groups. For immunohistochemistry, sections were subjected to heat-mediated antigen retrieval with 10 mM EDTA for 10 min at 95 °C. Endogenous peroxidases were blocked by 3% H₂O₂ and 10% methanol in Tris-buffered saline (TBS, 30 min at room temperature). Sections were permeabilized in 0.1% Triton X-100 for 15 min, blocked by a 30 min incubation in blocking buffer (TBS with 3% bovine serum albumin and 0.1% Triton X-100), followed by incubation at 4 °C with primary antibodies (diluted in blocking buffer) against the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) (WAKO rabbit; 1:500 dilution) and/or TREM2 (R&D Systems; sheep 1:500 dilution) for 2–3 days. After washing, sections were incubated with Alexa fluorophore-conjugated secondary antibodies (Invitrogen; anti-rabbit and anti-sheep) diluted 1:500 in blocking buffer for 1–2 days. To label amyloidogenic plaques, immunostained sections were incubated with 0.5% Thioflavin S (ThioS; Sigma-Aldrich) for 10 min and subsequently washed sequentially with 70% ethanol, 50% ethanol, and purified H₂0 before mounting on glass slides with VECTASHIELD® Antifade mounting media (Vector Labs).

Tissue sections (20 μm) were fixed with cold acetone for 5 min, rinsed three times for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The tissue sections were then free-floated in 20 mL of 1% thioflavin S (Thios) (Sigma, St. Louis, MO) aqueous solution for 5 min, followed by differentiation in 50% ethanol three times for 5 min each and washing in 1x PBS for 5 min. The sections were mounted with anti-fluorescence quenching sealing liquid. Images (4x magnification) were taken on the EVOS FL Auto Cell Imaging System and analysed using ImageJ. All images were preprocessed using the same threshold setting prior to analysis.