Cell culture and internalization assay
Raw 264.7 cells were kind gift from Prof. Liwu Li, they were maintained in DMEM containing 10% FBS at 37°C and 5% CO2. For internalization assay, RAW 264.7 cells transduced with lentivirus expressing GFP were plated on poly-lysine coated glass bottom tissue culture plate. 24 h later, cells were exposed to 2 μM MBSA-Texas red for 4 min at 37°C. Cells were imaged using an inverted Zeiss laser scanning microscope (LSM 880) with a 63X oil immersion lens. For pulse-chase experiments Raw 264.7 cells were plated on polylysine coated glass bottom tissue culture plate. 24 h later cells were pulsed with 2 μM MBSA-Texas red for 4 min washed and fresh media added. Cells were incubated at 37°C for 65 minutes, lysotracker was added to the cells and they were returned to the incubator for additional 25 min. Live cells were imaged using 63X oil immersion lens in an inverted Zeiss laser scanning microscope (LSM 880).
Texas-red maleylted BSA generation
BSA (Sigma) was incubated with NHS-Texas red (ThermoFisher) at pH 8.5 in a 1:3 ratio for half an hour at 4°C. Conjugation was stopped using Tris and the protein was dialyzed against PBS (phosphate buffered saline) exhaustively. The ratio of Texas red to BSA was found to be ~1:2.2. BSA-Texas red was then maleylated using maleic anhydride following a protocol described earlier; briefly maleic anhydride was added to BSA-Texas red with constant stirring, the pH was maintained above 8.5 using 5 N NaOH. This was followed by dialysis against PBS overnight. 10 mer polyG and polyA conjugated with FAM were acquired from IDT (Integrated DNA Technology).
Acute cortical slice experiment
All animal procedures were performed in accordance with the guidelines for the animal care of laboratory animals issued by Virginia Tech. The mouse strain used was C57Bl/6. 200 μm thick coronal brain slices from P35 mice (35 days old) were cut with a vibratome, the method was adapted from Wu and Hablitz, 2005. Slices were then incubated in a 24 well plate containing 5 μM MBSA-Texas Red (5 μM BSA-Texas Red as control) or 10 μM polyG (10 μM polyA as control) in 1 ml artificial cerebrospinal fluid (ACSF, Cold Spring Harbor Protocols) at 4°C with continuing oxygen supply for 30 min to allow binding. Afterward, oxygen supply was cut off and the plate was incubated at 37°C for 8 min to allow internalization. The brain slices were washed with phosphate buffered saline (PBS) 3 times and fixed with 4% paraformaldehyde (PFA). Neuronal marker NeuN antibody (Novus Biologicals, dilution ratio 1:150), astrocytic marker, GFAP antibody (Neuromab, 1;200) and microglial marker Iba1 antibody (Wako, 1:250) were used for immunostaining as needed. The slices were permeabilized overnight with 0.2% TritonX-100 in PBS, slices were blocked for 1 h in blocking buffer (0.2% TritonX-100 in PBS with 5% horse serum). Slices were incubated with NeuN antibody diluted in blocking buffer for 1 h followed by incubation for 30 min with Alexa 488 or Alexa 546 anti-Rabbit (Abcam) secondary antibody (1:500). Slices were then washed mounted on slides using Vectashield® with DAPI.