Donkey α mouse alexa fluor488

Immunostainings of brain sections and image acquisition were performed according to standard procedures as described earlier by Nordström et al. (2013) (link) and Herzer et al. (2015 (link), 2016 (link)). Blocking (RT, 1 h) and antibody incubations (4°C, o/n) occurred in 1%BSA/0.05% Triton-X/PBS. Primary antibodies were mouse-α-Aβ (6E10; 1:200, Covance), rabbit-α-Aβ₄₂ (1:50, Millipore), rabbit-α-GFP (1:50, Invitrogen), rabbit-α-Iba1 (1:200, WAKO), mouse-α-GFAP (1:200, Millipore), and oligomer-specific antibody rabbit-α-A11 (1:50, Invitrogen). Secondary antibodies used were donkey-α-mouse Alexa-Fluor488, donkey-α-rabbit Alexa-Fluor488, goat-α-rabbit Alexa-Fluor555, goat-α-mouse Alexa-Fluor555 (1:200, Invitrogen). For immunohistochemistry, subsequent staining was performed with biotin-α-mouse or biotin-α-rabbit (1:150, Jackson Immuno Research) and horseradish peroxidase streptavidin (1:200, Vector). For histochemistry, AEC reagent (BioGenex) was used. Coverslips were mounted with ProLongGold^® (Invitrogen) and subsequently analyzed by fluorescence or brightfield (Keyence) or confocal microscopy (Leica).

Standard fixation with 3.7% paraformaldehyde, followed by 0.1% Triton X permeabilisation and blocking with 0.25% BSA was performed prior to antibody incubations. Primary antibodies used were mouseαp16 JC8 (1:200), mouseα8‐oxoguanine (1:100, MAB3560 Millipore), rabbitαp21 (1:1,000, 12D1 Cell Signalling), rabbitαEGR2 (1:250, H220 Santa Cruz), goatαIL‐6 (1:100, AB‐206‐NA R&D Systems), followed by donkeyαmouse AlexaFluor‐488 or goatαrabbit AlexaFluor‐546 (1:500, Invitrogen), DAPI and Cell Mask Deep Red (1:10,000, Invitrogen). For 5‐bromo‐2′‐deoxyuridine (BrdU) assays, cells were cultured in 5 µM for 16 h prior to fixation. An additional DNA denaturation step with 4 M HCl for 10 min was performed following permeabilisation, and a conjugated mouseαBrdU‐AlexaFluor‐488 antibody (1:100, B35130 Invitrogen) used. Images were collected at 10X using the IN Cell 1000 microscope (GE) and the Developer analysis software (GE) was used for image analysis as described previously (Bishop et al., 2010).
Please also see Appendix S1.