Trueblack lipofuscin autofluorescence quencher

Human donor eyes were obtained through the Foundation for Fighting Blindness Eye Donor Program (Columbia, Maryland, USA). Donor eyes were not clinically diagnosed with a retinal degeneration. Human retinal tissues were fixed with 4% PFA and 0.5% glutaraldehyde in Dulbecco’s PBS (D-PBS). Pieces of retina-RPE-choroid were then fixed in 4%PFA, infiltrated with 10%–20% sucrose in PBS, incubated in SD-OCT medium, placed in base molds filled with SD-OCT, frozen rapidly in liquid nitrogen, and kept at −80°C until processed. Cryosections (10 μm) were collected on an HM 505E cryostat (Microm) equipped with a CryoJane Tape-Transfer system (Leica). In brief, slides were pretreated with citrate buffer (pH 6.0; MilliporeSigma) in a microwave for 2 minutes, followed by blocking with 0.1% Triton X-100 and 10% donkey serum in PBS for 2 hours. The slides were then incubated with the primary Ab (mouse anti-CDCP1, 9A2; dilution, 1:500) in PBS with 0.1% Triton X-100 and 2% donkey serum overnight at room temperature, washed 3 times in PBS, and incubated with the secondary Ab (FITC-conjugated anti-mouse IgG, Jackson ImmunoResearch; dilution, 1:500) for 2 hours. The sections were mounted with Fluoromount-G (Abcam) and treated with TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) to suppress tissue autofluorescence. Images were taken using a BZ-X700 microscope and SP8 confocal microscope.

Left atrial appendage specimens were fixed in 4% paraformaldehyde, frozen in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and cut into 8 μm thick slices. Sections were blocked with ImmunoBlock (KAC Co., Ltd. Kyoto, Japan) for 1 hour and then treated with TrueBlack lipofuscin autofluorescence quencher (BioTium, Fremont, CA, USA) for 30 seconds before incubation in primary antibodies for CD68 (1:50 dilution, clone PG-M1, Dako, Glostrup, Denmark) and CCR2 (1:100 dilution, clone 7A7, Abcam, Cambridge, UK) overnight at 4°C. Fluorescent-conjugated secondary antibodies (Alexa Fluor 488 anti-mouse IgG3, 1:1000 dilution, Alexa Fluor 594 anti-mouse IgG2a, 1:1000 dilution, Thermo Fisher Scientific) were applied and then the cells were incubated for 2 hours at room temperature. Sections were finally incubated with DAPI (1 μg/ml, Thermo Fisher Scientific), washed with PBS and mounted with Fluoromount-G (Thermo Fisher Scientific). Slides were examined using and LSM880 confocal microscope (Carl Zeiss, Oberkochen, Germany). The number of CD68-positive cells (monocytes/macrophages) and CCR2-positive monocytes/macrophages in 10 random field of view images (20x objective) was counted manually in a blinded fashion.

Lipofuscin was quenched with TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, Fremont, CA). Briefly, vibratome sections were washed once with PBS and treated for 30 s with TrueBlack 1 × prepared in 70% ethanol. Finally, sections were washed three times with PBS and then immunostained as described above.

Fixed tissues from uninfected and HIV- or SIV-infected brains were paraffin-embedded, followed by sectioning and mounting. Slides were rehydrated and subjected to antigen retrieval by boiling in 10 mM sodium citrate, pH 6, before immunostaining. Tissues were labeled with primary antibodies (anti-Iba-1 [Abcam], anti-hCD3ε [Millipore], anti-HIV-1 p24 and anti-SIV p27] overnight at 4°C. Autoflourescence was quenched using a TrueBlack lipofuscin autofluorescence quencher (Biotium), followed by application of appropriate secondary antibodies (53 (link), 54 (link)). Images were acquired on a Wave Fx (Quorum Technologies) spinning disk confocal microscope using Volocity (Perkin Elmer) acquisition and analysis software.

Human/murine phospho-tau pSer202/Thr205 (AT8, Cat# MN1020), Tau46 (Cat# 13-6400), Alexa Fluor dye-labeled cross-absorbed donkey secondary antibodies, and TRIzol RNA isolation reagent (Cat# 15596026) were purchased from ThermoFisher Scientific. Goat anti-Olig2 (Cat# AF2418) and GAD1 (Cat# AF2086) polyclonal antibody were purchased from R&D Systems. Rabbit anti-GFAP (Cat# G9269), WFS1 (Cat# 1158-1-AP), and Aβ (Cat# Ab2539) polyclonal antibodies were purchased from Sigma-Aldrich, Proteintech, Abcam, and DAKO, respectively. Rat anti-P2RY12 (Cat# 848002) was purchased from BioLegend. RNAscope HiPlex Ancillary Kit (Cat# 324120) and human-specific RNA probes were purchased from Advanced Cell Diagnostics. TrueBlack lipofuscin autofluorescence quencher (Cat# 23007) was purchased from Biotium. Fluoromount-G Mounting Medium (Cat# 0100-01) was purchased from SouthernBiotech.

A 1-in-6 series of coronal forebrain hemisections and a 1-in-48 series of 40 μm coronal spinal cord sections from each mouse were stained on slides using a modified immunofluorescence protocol using the TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) (19 (link), 63 (link)). See Supplemental Methods for further details, including primary and secondary antibodies. To visualize GABAergic interneurons for stereological counts, a 1-in-6 series of coronal hemisections were stained on slides using a modified immunoperoxidase protocol (18 (link), 50 (link)). See Supplemental Methods for further details, including used antibodies. All images were taken on a Zeiss Axio Imager.Z1 microscope using StereoInvestigator (MBF Bioscience) software or a Zeiss LSM880 confocal laser scanning microscope with Airyscan and ZEN 2 software (blue edition, Zeiss).