Ab150129

Immunohistochemistry was performed as previously described^{27 (link),43 (link),44 (link)}. Specimens were incubated with goat anti-C. trachomatis/C. pneumoniae (this antibody is used to detect both of these Chlamydia species; Abcam ab20929; 1:400) and/or rabbit anti-Aβ peptide (Abcam ab201060,1:500). Secondary antibodies were donkey anti-goat Alexa Fluor 488 (Abcam ab150129 1:400), donkey anti-rabbit 647 (Invitrogen A31573; 1:500). Antibodies were diluted in blocking buffer (2% bovine serum albumin with 0.3% Triton X-100 in PBS). Cryostat sections were first incubated with blocking buffer for 60 min at room temperature, followed by overnight incubation with primary antibodies at 4 °C. Sections were washed for 3 × 5 min, then incubated with secondary antibodies for 1 h. Cell nuclei were stained with 4′6-diamidino-2-pheylindole (DAPI).

NCSCs (Neural Crest Stem Cells) were grown in culture slides until 80% confluency. Cells were fixed using 4% Paraformaldehyde for 20 min at room temperature. After 3 steps of washing with 1× PBS, cells were permeabilized using 0.2% Triton X-100 for 15 min at 37 °C and washed three times with 1× PBS. Non-specific binding was blocked using 0.5% FBS in 1× PBS for 1 h at room temperature. Antibodies against P75 (Millipore 07-476, 1/200, Darmstadt, Germany), SOX10 (R&D Systems MAB2854, 1/200, Minneapolis, MN, USA), Nestin (Chemicon #MAB5326, 1/500, Darmstadt, Germany), and AP2a (Invitrogen #MA1-872, 1/100, Waltham, MA USA) were incubated overnight at 4 °C with gentle shaking. After three washing steps with 1× PBS, slides were incubated with secondary antibodies (Donkey Anti-Rabbit Alexa Fluor^® 647, Abcam ab150075, 1/1000, Donkey Anti-Goat Alexa Fluor^® 488, Abcam ab150129, 1/1000, Goat anti-Mouse AF 488, Invitrogen #A-11001, 1/500) for 1 h at room temperature. After washing three times with 1× PBS, slides were mounted in VECTASHIELD^® Antifade Mounting Medium with DAPI to counter-stain nuclei. Images were acquired using a Widefield fluorescence microscope with optical sectioning Zeiss Apotome.2 (Zeiss, Oberkochen, Germany).