Sucrose (#S0389), Chloroquine (#C6628), Quinine (#6119–47-7), Tris base (#11,814,273,001), Glycine (#G8898), SDS (#L3771), Triton™ X-100 (#T9284), EDTA (#E9884), EGTA (#3889), Dimethyl sulfoxide (#D8418), Thio-T (#T3516), HHT (#SML1091), Emitine (#E2375) were purchased from Sigma-Aldrich. Anisomycin (#S7409) was purchased from SelleckChem. DMEM, high glucose, GlutaMAX Supplement (#10,566,016) was purchased from GIBCO. Complete™, Mini, EDTA-free Protease Inhibitor Cocktail (#11,836,170,001) was purchased form Roche. Lipofectamine 3000 (#L3000015), Lipofectamine RNAi-MAC (#13,778,150), Puromycin (ant-pr-1) were purchased from Invitrogen. Pierce 16% Formaldehyde (w/v), Methanol-free (#28,908), RPMI-1640 (#11,845,085), RNAse A (#12,091,021) were purchased from ThermoFisher.
Drosophila genetics and husbandry
We used wild type w1118 as control. We obtained the following lines from the Bloomington Drosophila Stock Center: elav-GAL4 (B8765), UAS-APP.C99 (B33783), UAS-APP (B6700), UAS-APP;UAS-BACE (B33798), UAS-Rab5-GFP (B43336), UAS-KDEL-GFP (B9898), UAS-LAMP1-GFP (B42714), UAS-Rab5-WT (B43336), UAS-Rab5-CA (B43335), UAS-Rab5-DN (B9772), UAS-Fip200 (B91226), UAS-ABCE1-RNAi (B31601), UAS-Lcp2-C99 (B33785), UAS-ZNF598-RNAi (B61288), UAS-Ltn1 (B30116). We obtained UAS-Clbn-RNAi (v103351) from Vienna Drosophila Stock Center. We obtained UAS-ABCE1 (F001097), UAS-ZNF598 (F001909), UAS-Pelo (F003036), UAS-Vms1 (F002500) from FLYORF. T. Littleton provided MHC-GAL4, S. Birman provided TH-GAL4, F. Kawasaki provided UAS-mito-GCaMP, N. Bonini provided UAS-HspA1L, Eric Baehrecke provided UAS-ATG1, Paul Taylor provided UAS-VCP. The indicated UAS RNAi and OE fly lines were crossed to Mhc-Gal4 or elav-Gal4 driver lines for muscle or pan-neuronal expression, respectively. Fly culture and crosses were performed according to standard procedures. Adult flies were generally raised at 25 °C and with 12/12 h dark/light cycles. Fly food was prepared with a standard receipt (Water, 17 L; Agar, 93 g; Cornmeal, 1,716 g; Brewer’s yeast extract, 310 g; Sucrose, 517 g; Dextrose, 1033 g).
10–20 male flies were transferred to a clean empty food vial. The flies were then allowed to get accustomed to the new environment for 3–4 min and subsequently measured for bang-induced vertical climbing distance. The performed was scored as percentage of flies crossing the 8 cm mark within 12 s. Each experiment was carried out ≥ 4 times.
Flies were reared in vials containing cornmeal medium. Flies were anesthetized using CO2 and collected at a density of 20 male flies/vials. All flies were kept at humidified, 12 h on/off light cycle at 25 °C. Flies were flipped into fresh vial every 3 days and scored for death. Each set of experiment was carried out ≥ 4 times.
Extraction of fly proteins for western blot analysis
Around 5 fly thoraces were homogenized in 80 µl of either regular lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X100, protease inhibitors) or Urea buffer (6 M Urea, 50 mM Tris–HCl, 150 mM NaCl, 0.1% Triton X100, protease inhibitors) on ice. Each sample was homogenized by a mechanical homogenizer for 30 secs. The homogenized samples were incubated on ice for 30 min before centrifuging at 15,000 rpm for 20 min at 4 °C. 30 µl of supernatant was mixed with 10 µl of 4 × Lammaelli buffer (BioRad #161–0747) and boiled for 5 min at 100 °C. The protein lysate was cooled, centrifuged and loaded onto 4–12% Bis–Tris gel (Invitrogen #NP0321) or 16% Tricine gel (Invitrogen #EC66955) with 1 × MES (Invitrogen #NP0002) as running buffer.
Aversive taste memory
Taste memory assay was performed as described previously , with slight modifications. Briefly, one-week old flies were starved for 12–18 h in an empty vial on wet Kimwipe paper. Flies were later anesthetized using ice and fixed on a glass slides by applying nail polish to their wings. 10–15 flies were used for each set of experiment. The flies were then incubated in a humid chamber for 2 h to allow them to recover from the procedure. The experiment was divided into three phases. The first phase is the pretest, when the flies were presented with 500 mM sucrose stimuli (attractive tastant) to their legs using a Kimwipe wick. Flies that showed positive proboscis extension to the stimulus were used for the next phases. The second phase was the training phase, when the flies were presented with 500 mM sucrose stimuli at their legs while simultaneously being punished by applying 10 mM quinine (aversive tastant) on their extended proboscis. Training was repeated 15 times for each fly. The last phase is the test phase when the flies were given 500 mM sucrose to their legs at different time intervals (0, 5, 15, 30, 45, and 60 min), and the proboscis extension response was recorded. Each experiment was carried out ≥ 4 times.
We performed immunostaining analysis of adult fly muscle as previously described . Briefly, fly thoraxes were dissected and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710) in phosphate buffered saline and 0.3% Triton X-100 (PBS-T). The tissues were then washed three times with PBS-T. The samples were incubated for 30 min at room temperature in blocking buffer: 0.5% goat serum in PBS-T. The indicated primary antibodies (anti-Ubiquitin, Abcam ab140601, 1:1000; anti-Rab5, Abcam ab31261, 1:1000; anti-P62, Abcam ab178440, 1:1000; Anti-DsRed, clontech #632496, 1:1000; 6E10, Biolegend #803001, 1:1000; anti-LAMP1, DSHB 1D4B, 1:100) were added and samples were incubated overnight at 4 °C. The samples were washed three times with PBS-T and subsequently incubated with the indicated secondary antibodies (Alexa Flour 488 (A32723), Alexa flour 594 (A11036), Invitrogen, 1:200) for 4 h at 4 °C. The tissues were washed three times with PBS-T and mounted in slow fade gold buffer (Invitrogen).
Immunostaining of adult brains were performed as previously described . Briefly, we dissected the brain tissues of adult flies and fixed them on ice for 30–45 min in fixing buffer (940 µl of 1% PBS-T and 60 µl of 37% formaldehyde). The tissues were washed three times in 0.1% PBS-T and blocked overnight at 4 °C in blocking buffer (1 ml 1 × PBS, 0.1% Triton-X, 5 mg/ml BSA). The tissues were incubated for 16 h at 4 °C with the indicated primary antibodies (anti-TH, Pel-Freez P40101-150, 1:1000; anti-mcherry Abcam ab167453, 1:1000; anti-Ubiquitin, Abcam ab140601, 1:1000; 6E10, Biolegend #803,001, 1:1000; anti-Rab5, Abcam ab31261, 1:1000). Samples were washed three times with 0.1% PBS-T and subsequently incubated with the appropriate secondary antibodies (Alexa Flour 488 (A32723), Alexa flour 594 (A11036), Invitrogen, 1:200) for 4 h at 4 °C. Samples were mounted in slow fade gold buffer (Invitrogen) and viewed using a Leica SP8 confocal microscope.
The Flag-C99 plasmid was generated by inserting C99 coding sequence with stop codon to the pCMV-Flag-Myc vector via the NotI/BamHI cloning sites. The following PCR primers were used to amplify C99 sequence from pCAX-C99:
No ER Flag-C99-myc was generated by deletion of the TGA stop codon from Flag-C99 using the following PCR primers:
ER-Flag-C99 was generated by inserting the ER-target sequence of human APP upstream and in-frame with Flag-C99 using the following PCR primers:
ER-Flag-C99-myc was generated by deletion of the TGA stop codon from ER-Flag-C99 using the following PCR primers:
ER-C99 was generated by deleting the Flag sequence from ER-Flag-C99 using the following PCR primers:
ER-C99-VVE-HA was generated by inserting the HA epitope into ER-C99 using the following PCR primers:
ER-C99-SK-HA was generated by inserting the HA epitope into ER-C99 using the following PCR primers:
ER-C99-YK-HA was generated by inserting the HA epitope into ER-C99 using the following PCR primers:
Ifn-C99 was generated by substituting the human APP ER-targeting sequence in ER-C99 with the signal sequence of Ifn using the following PCR primers:
Opn-C99 was generated by substituting the human APP ER-targeting sequence in ER-C99 with the signal sequence of Opn using the following PCR primers:
ER-C99-3 K-A was generated by mutating the three K residues in ER-C99 immediately C-terminal of the TM domain of C99 with three A residues using the following PCR primers:
HeLa cells and HEK293T cells were purchased from ATCC. HEK293T cells stably transfected with Aβ42-YFP were described before . Cells were cultured under standard tissue culture conditions (1 × DMEM medium, GIBCO, 10% FBS, 5% CO2, 37 °C).
Protein extraction from cultured cells and western blotting
HeLa cells were transfected with the respective plasmids. Cells were washed with 1X PBS 30 h post transfection and lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X100, protease inhibitors). The cells were centrifuged at 13,000 rpm for 20 min at 4 °C. Protein analysis was carried out by Bradford method. The supernatant was then mixed with 4 × protein loading buffer and loaded onto either 4–12% bis–tris gels using MES as running buffer or on 16% Tricine gel and immunoblotted onto PVDF membranes. The membranes were blocked with blocking buffer (5% BSA in TBST) and incubated with following primary antibodies (Anti-Flag, Sigma-Aldrich F1804, 1:2000; Anti-GFP, ProteinTech 66,002, 1:1000; Anti-ubiquitin, Abcam ab140601, 1:1000; Anti-Actin, Sigma-Aldrich A2228, 1:500; 6E10, Bio Legend 803,001, 1:1000; Anti-Myc, ProteinTech 16,286, 1:1000; Anti-APP C-term (C1/6.1), ThermoFisher 512,700, 1:500; Anti-Calnexin, ProteinTech 1047–2-AP, 1:1000; Anti-RPL22, ProteinTech 25,002-1-AP, 1:1000; Anti-Sec61B, ProteinTech 15,087-1-AP, 1:2000; Anti-HA, Sigma-Aldrich 12CA5, 1:3000; Anti-eRF1, Cell Signaling 13,916, 1:1000; Anti-eRF3, Cell Signaling 14,980, 1:1000; Anti-DDX19B, ProteinTech 18,285-1-AP, 1:1000; Anti-ZNF598, GeneTex GTX119245, 1:250; Anti-ANKZF1, ProteinTech 20,447-1-AP, 1:1000; Anti-NEMF, ProteinTech 11,840-1-AP, 1:500; Anti-Ltn1, ProteinTech 28,452-1-AP, 1:1000; Anti-RPL7a, ProteinTech 15,340-1-AP, 1:1000; Anti-Rack1, Santa Cruz sc-17754, 1:1000; Anti-PSD95, Abcam ab18258, 1:1000). Goat anti-Rabbit IgG HRP, Santa Cruz sc2004 or Goat anti-Mouse IgG-HRP, Santa Cruz sc2005 antibodies were used for detection at 1:10,000 dilution. For data quantification of western blots, signal intensity was measured and calculated using NIH Image J.
Special steps were taken during SDS PAGE to better resolve the different APP.C99 species. We tried to resolve the gel as much as possible such that the lower protein ladder (10–25) was distinctly separated. This often required running the gel front until it was just at the bottom of the gel. Care was taken not to run the ~ 10 KD protein ladder band out as the C99 lower band was just beyond this molecular weight. The gel was transferred onto PVDF membrane using regular 1 × Transfer buffer. The transfer conditions were 350 mA constant for 45 min for the bis–tris gel or 85 V constant for 1 h.
Membrane fraction isolation
To isolate a crude membrane fraction containing ER, lysosomes and Golgi, cells were washed with 1 × PBS before incubating with 50 mM Tris–Cl, 150 mM NaCl, 25 mg/ml digitonin for 10 min at 4 °C. The cells were then centrifuged at 2000 rpm for 10 min. The lysate was then incubated with buffer containing 1% Triton X100 to solubilize the membrane structures and incubated at 4 °C for 30 min. This was followed by centrifugation at 7000 rpm for 10 min to pellet the nuclei and supernatant containing membrane protein extract.
Cells were washed with 1 × PBS 30 h post transfection and lysed using either lysis buffer or 6 M Urea buffer (6 M urea, 50 mM Tris–HCL, 150 mM NaCl, protease inhibitors) as indicated. 5% of the lysate was kept aside to load as input. The remaining lysate was incubated with the desired antibody at 4 °C overnight. If using urea buffer, the lysate was diluted to 0.5 M urea before incubating with the desired antibody (4 µl of 6E10 (Bio Legend), 2 µl for Sec61B (ProteinTech), and 2 µl for Rpl22 (ProteinTech) antibodies). The following day protein A/G beads (Santa Cruz sc2003) were added to the antibody-lysate mixture and incubated for 2hs at 4 °C. The beads were then washed with lysis buffer three times and the wash was removed by centrifugation. The beads were suspended in 4 × SDS loading buffer and boiled for 5 min before loading onto bis–tris gels.
Immunostaining of cultured cells
Cells on coverslips were washed with PBS twice and fixed in 4% paraformaldehyde/PBS solution for 15 min at RT. Cells were washed repeatedly with 1 × PBS prior to incubating with PBS containing 0.1% Triton X-100 for 20 min. Cells were then incubated in blocking buffer (1 × PBS, 0.1% Triton X100, 2% BSA) for 30 min. After blocking, desired primary antibodies (Anti-Rab5, Abcam ab31261; Anti-LC3, ProteinTech 14,600-1-AP; 6E10, BioLegend 803,001; Anti-Calnexin, ProteinTech 10,427-2-AP; Anti-Puromycin, Millipore MABE343; M78, Gift from Dr. Glabe) were added to the blocking buffer at 1:1000 concentration and cells were incubated with the antibody solution overnight. After washing with 1 × PBS the following day, cells were incubated in appropriate secondary antibodies for 1 h. Cells were washed again and the coverslips were mounted on slides using DAPI-containing mounting medium.
HeLa cells were plated one day before transfection with plasmids and siRNAs as noted in the text. The medium was changed after overnight transfection. 30 h post transfections, the DMEM containing 0.5 mg/ml MTT reagent (Sigma M2128) was added to the cells. The cells were incubated at 37 °C for 120 min, and the medium was removed following which the purple formazan crystals were dissolved using DMSO. After shaking on a shaker for 30 min to ensure complete dissolution of the crystals, the resulting absorbance was measured at 570 nm.
Plasmid transfections and siRNA knockdown
Cell transfections were performed by using Lipofectamine 3000 (cat#: L3000015, Invitrogen), and siRNA knockdown experiments were performed using Lipofectamine RNAiMAX reagent (cat#: 13,778,150, Invitrogen), according to manufacturer’s instructions. Different plasmids were used for transfection of HeLa and HEK293T cells using lipofectamine 3000 following instructions from the manufacturer. Briefly, cells were plated at 70% confluency the day before in DMEM without antibiotics. On the day of transfection, plasmid DNA and lipofectamine reagent were individually mixed in OptiMEM and then mixed together such that the plasmid/reagent = 1:3. After incubation of 10 min at room temperature, the plasmid-reagent mixture was added dropwise to the cells. Medium was changed the following day and cells were analyzed 30 h post transfection. For siRNA treatments, a similar protocol was followed with the final concentration of siRNA at 450 pmol for a 10 cm dish (for a 10 cm dish: 10 µg of DNA and 20 µl of Lipofectamine 3000, for 6 cm dish: 3–5 µg of DNA and 5 µl of Lipofectamine 3000 were added). For siRNA and plasmid co-transfection, the cells were first treated with siRNA. 24 h post transfection with siRNA, the medium was replaced and plasmid DNA transfection was carried out.
Translocon capture with ConA beads
Proteins were extracted from cells grown on 10 cm culture dishes. Proteins were extracted as indicated either in Urea buffer or crude membrane preparations. This was followed by incubating for 2hs at 4 °C with 50 µl of Con A beads (Sigma-Aldrich GE17-0440–03). The beads were washed with lysis buffer three times before adding the 4X SDS loading buffer. For RNAse A and EDTA treatments, membrane fractions were isolated from HeLa cells transfected with APP.C99 as described before followed by capture with ConA beads. After washing the beads as described before, the beads were centrifuged and RNaseA (1 µg/ml) and EDTA (50 mM) were added to the beads. RNAse A treatment was incubated at 37 °C for 30 min while EDTA treatment was left on ice for 30 min. An aliquot of untreated beads was included as control. After the treatment period, the beads were again centrifuged. Supernatants and beads were then separated and mixed with SDS loading buffer, boiled and loaded onto 4–12% Bis–Tris gel.
Puromycin labeling of ribosome stalled newly synthesized proteins
At ~ 30 h post transfection, HeLa cells were incubated with fresh DMEM medium containing HHT (5 µM) for 10 min at 37 °C. Emetine (100 µM) and Puromycin (100 µM) were then added to the medium and cells were incubated further for 15 min at 37 °C. Cells were then washed and harvested in lysis buffer, following which proteins were extracted as previously described.
Puromycin labeling of stalled proteins: Puromycin labeling was done as described , with slight modifications. Hela cells were seeded on coverslips in a 6-well plate and transfected with pCAX-C99 (Addgene #30,146). After 48 h transfection, cells were treated with HHT for 5 min, thereafter Puromycin (50 µg) and Emetine (100 µg) were added and cells were incubated for 5–7 min. Once the incubation was over, cells were permeabilized by 0.02% digitonin in Permeabilization buffer (50 mM Tris–HCl, pH7.5, 5 mM MgCl2, 25 mM KCl, 355 mM cyclohexamide, 10 units RNAseOut and 0.02% digitonin) for 2 min. Permeabilized cells were washed twice with washing buffer (permeabilization buffer without digitonin) and fixed in 4% paraformaldehyde for 30 min. The Permeabilization and washing steps were performed in ice-old buffers. Cells were then stained with the mOC78 and Puromycin antibody and observed under the confocal microscope.
HeLa cells were treated with the following drug concentration and times as indicated in the main text. Cycloheximide: 50 µg/ml for 4 h; HHT: 5 µM for 10 min; emetine: 100 µM for 15 min; Puromycin: 100 µM for 15 min.
Aβ42-YFP aggregation assays
Aβ42-YFP cells were described before . The pCAX-C99 (Addgene #30,146) plasmid was transfected into Aβ42-YFP cells using Lipofectamine 3000 according to the Manufacturer’s manual. 48 h after transfection, YFP signal was detected under confocal microscope. For siRNA knockdown experiments, Aβ42-YFP cells at 50% confluence were transfected with siRNA targeting ANKZF1 (Invitrogen HSS113541), ABCE1 (Invitrogen HSS109285), VCP (Invitrogen HSS123962), NEMF (Invitrogen HSS113541) and ZNF598 (Invitrogen HSS132049) for 24 h using RNAiMAX (Invitrogen #13778150), according to the manufacturer's instructions. Then cells were transfected with pCAX-C99 plasmid for further 48 h. The cells were washed with 1 × PBS and fixed with 4% paraformaldehyde for 15 min. YFP signals were acquired using confocal microscope (Leica, SP8).
For immunostaining assays, Aβ42-YFP cells were transfected with ER-FLAG-C99 and FLAG-C99 respectively for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min. After blocking with 5% BSA for 1 h, cells were incubated with anti-FLAG antibody (Sigma-Aldrich, F7425) at room temperature for 2 h. Slides were washed three times with PBS and incubated with Alexa Fluor 633-conjugated secondary antibodies for 1 h at room temperature. Images were acquired using confocal microscope (Leica, SP8).
B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax mice (5xFAD mice) were purchased from Jackson Laboratory. The mice were bred and housed in the Stanford University animal facility. All mouse experiment-related protocols were approved by the Stanford’s University School of Medicine’s APLAC committee.
Lenti-scramble control shRNA viral particles expressing GFP (lenti-GFP) and mouse ZNF598 shRNA lentiviral particles expressing GFP (Lenti-shZNF598) were purchased from OriGene (CAT#: TL513709V). Viral titers were > 10E7 TU/ml. Neonatal pups were injected with either Lenti-GFP or Lenti-shZNF598 at P0/P1 stage as described before . Briefly, pups were cryo-anaesthetized before injection. Following cessation of any movement, the pups were placed side up and 2 µl of virus was injected on each side with a 32 gauge needle (BD catalogue #324,909). The injection site is about 2/5th of the distance between the eye and the lambda intersection as described before . Following injection, the pups were warmed and returned to the cage with the mother for further care. Mice were sacrificed after 5 months. Brain samples were dissected and subjected to fixation with 4% PFA and later treatment with 10% and 20% sucrose for 24 h. Frozen sectioning was performed according to Stanford Pathology facility protocols. Subsequent immunostaining was carried out by following standard procedures.
Synaptosome isolation from mouse brain
Mouse brain tissue was homogenized in cold lysis buffer (0.32 M sucrose, 5 mM HEPES, complete protease and phosphatase inhibitors) at 5:1 buffer to tissue (mg) volume using a mechanical homogenizer for 30 s at 800 rpm. The resulting homogenate was centrifuged at 1000 g at 4 °C for 10 min to separate the nuclei pellet. The supernatant was further centrifuged at 12000 g for 10 min at 4 °C to pellet the synaptosomes. The pellet was further suspended in 1 × TBS solution and used for SDS gel electrophoresis. The purity of the synaptosomal isolation was checked by detection of PSD95 protein in the pellet but not supernatant fraction.
Mouse brain frozen section immunofluorescence staining
Animals were sacrificed by cervical dislocation and subsequently harvested followed by storing in 4% formalin (Sigma) for 24 h. Tissues were then moved into 10% sucrose solution for 24 h and 20% sucrose for 24 h sequentially. After processing, tissues were embedded with OCT. Ten-micrometer sections were fixed in cold acetone for 10 min and then air dried at room temperature for 1 h. In order to remove OCT, slides were placed in 1 × PBS wash buffer for 10 min followed by processing with 0.3% PBST. After washing with 1xPBS, excess wash buffer was wiped off from slide without drying sections. Tissues were blocked with 5% goat serum for 1 h at room temperature followed by incubating with primary antibodies (Anti-IBA1, Novus NBP2-19,019; Anti-ZNF598, Genetex GTX119245; Anti-Rack1, Santa Cruz sc-17754; MOAB2, Novus NBP2-13,075; Anti-CTSD, ProteinTech 55,021-1-AP; Anti-Lamp1, DSHB 1D4B; Anti-RPL22, ProteinTech 25,002–1-AP) overnight at 4 °C. Samples were rinsed in wash buffer for 5 min. After wiping off excess buffer, samples were incubated with secondary antibodies (conjugated to fluorophores) in the dark for 2 h at room temperature. After washing with PBS for 3 times, samples were ready for mounting with mounting media. Images were taken on a Leica SP8 confocal microscope.
Immunostaining for Cathepsin D (Abcam), 6E10 (Bio Legend), Rab5 (Abcam), LAMP1 (DSHB), P62 (Abcam), and IBA-1 (Novus) were performed on frozen brain tissue sections (5 μm). Briefly, the frozen sections were washed in 1 × PBS, and then permeabilized with 0.5% Triton X-100 for 15 min, blocked with 3% goat serum. Slides were incubated with anti-Cathepsin D, 6E10, Rab5, LAMP1, P62, IBA-1 antibody overnight at 4 °C. Slides were washed three times with PBS and incubated with Alexa Fluor 488- and 569-conjugated secondary antibodies for 1 h at room temperature. Slides were washed three times with PBS and mounted. Images were acquired using confocal microscope (Leica, SP8). The number of positive dots labeled by Cathepsin D or LAMP1 were counted in each cell.
Human tissue samples from patients with AD and controls were provided by the UCSF Neurodegenerative Disease Brain Bank. Demographic information is provided in Supplementary Table 1. Informed consent to undergo autopsy was provided by patients and/or their surrogates, following the principles outlined in the Declaration of Helsinki. Formalin-fixed, paraffin-embedded tissue blocks from the inferior temporal gyrus and/or middle frontal gyrus were cut into 8-microns thick sections and mounted on glass slides.
Paraffin section immunofluorescence staining of human brain samples
AD and control human brain sections were deparaffinized in xylene and rehydrated through graded ethanol series. Antigen retrieval was performed by incubating the slides in a steamer with citrate buffer (pH 6.0) (Sigma) for 30 min. The slides were then washed for 10 min in 1 × PBS and incubated in 3% H2O2 (in methanol) for 10 min in order to block any endogenous peroxidase activity. Slides were then washed with 1 × PBS for 10 min. To prevent nonspecific binding and excessive background, slides were blocked with a serum-free buffer for 30 min. Primary antibodies (Anti-Rack1, Santa Cruz sc-17754, 1:500; Anti-ZNF598, GeneTex GRX119245, 1:250; Anti-ABCE1, Gift from Dr. Hegde; Anti-NEMF, ProteinTech 11,840-AP, 1:500; Anti-RPL22, ProteinTech 25,002-1-AP, 1:1000; Anti-ANKZF1, ProteinTech 20,447–1-AP, 1:1000) were applied on the slides and incubated overnight at 4 °C. Slides were washed with PBS and incubated with secondary antibody (conjugated to fluorophores) for 2 h at room temperature. After washing with 1 × PBS, mounting medium was used to mount the slides. Images were taken by Leica SP8 microscope.
Quantification and statistical analysis
Statistical analysis was performed using GraphPad Prism 8 (Windows version 8, GraphPad Software, San Diego, CA, USA). Student’s t test and one-way ANOVA test with Tukey’s post hoc test were used for statistical evaluation. All data are represented as mean ± S.D, with P < 0.05 being considered statistically significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.