Mouse brain sections (10 μm) were mounted on collagen-coated glass slides (Thermo Scientific), fixed in acetone solution for 10 min, washed in Tris-buffered saline and then exposed to methanol for 5 min. Nonspecific labelling was prevented by incubating the sections in 5% bovine serum albumin (Sigma-Aldrich) for 1 h 30 min before incubating for 16 h at 4 °C with the following specific primary antibodies (1:500 dilutions): Aβ42 (BioLegend, San Diego, CA, USA). The sections were washed 3 times (5 min each) with phosphate-buffered saline with 0.1% Tween 20 and then incubated for 1 h in the dark with specific fluorescent secondary antibodies (1:500; Invitrogen). All brain sections were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and visualised with a confocal microscope (Carl Zeiss, Oberkochen, Germany).
A confocal microscope
Manufactured by Zeiss
Sourced in Germany, United States, Japan
A confocal microscope is an optical imaging technique used to increase the optical resolution and contrast of a micrograph by using a spatial pinhole to block out-of-focus light. It allows for three-dimensional imaging of a specimen by scanning it point-by-point and reconstructing the image.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Fluo 4 am, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Fluorescent in situ hybridization kit, by RiboBio (3 mentions)
Rna fish kit, by RiboBio (3 mentions)
Glass culture slides, by BD (3 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (3 mentions)
Brdu labeling reagent, by Thermo Fisher Scientific (2 mentions)
Pkh67, by Merck Group (2 mentions)
Freezing microtome, by Leica camera (2 mentions)
Antibody diluent, by Agilent Technologies (2 mentions)
8 well chamber slide, by BD (2 mentions)
Fish kit, by RiboBio (2 mentions)
Rabbit antihuman galectin 1, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Rhodamine red, by Thermo Fisher Scientific (2 mentions)
242 protocols using a confocal microscope
1
Immunohistochemical Analysis of Amyloid-β in Mouse Brain
2
Quantifying Intracellular ROS Levels
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ROS generation was assessed in cells treated with imatinib for 30 or 60 min. Intracellular ROS levels were determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which oxidizes within cells to fluorescent dichlorofluorescein. Control and imatinib-treated cells were incubated with 100 µM DCFH-DA for 30 min at 37°C.
Next, the cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were co-stained with 2 µM 4′,6-diamidino-2-phenylindole (Molecular Probes; Thermo Fisher Scientific, Inc.) at 37°C. After washing three times with PBS, the cells were mounted using VECTASHIELD mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA), and immunofluorescence was detected using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Then, MEF of MEF CHOP−/− cells were stained with 100 µM DCFH-DA for 30 min at 37°C. The cells were then transferred to FACS tubes and analyzed using the FL1 channel on a FACScan cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Next, the cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were co-stained with 2 µM 4′,6-diamidino-2-phenylindole (Molecular Probes; Thermo Fisher Scientific, Inc.) at 37°C. After washing three times with PBS, the cells were mounted using VECTASHIELD mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA), and immunofluorescence was detected using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Then, MEF of MEF CHOP−/− cells were stained with 100 µM DCFH-DA for 30 min at 37°C. The cells were then transferred to FACS tubes and analyzed using the FL1 channel on a FACScan cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
3
Quantifying Intracellular ROS Levels
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ROS levels were determined using carboxy-DCF-DA (2ʹ,7ʹ–dichlorofluorescin diacetate) (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated for 30 min with 20 µM carboxy-DCF-DA, and then assessed by fluorescence-activated cell sorting (FACS) or a confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
4
Paraformaldehyde Fixation and DAPI Staining
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Cells were fixed by 4% paraformaldehyde (Aladdin, Shanghai, China) for 10 min at room temperature, then rinsed by PBS thrice, and permeabilized by 0.3% Triton X‐100 (Aladdin) for 10 min. Thereafter, cells were rinsed by PBS thrice, next stained with DAPI (Meilune Biotech) for 15 min. At last, cells were captured by a confocal microscope (Zeiss).
5
Assessment of Proliferative S-Phase Cells
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To assess proliferative S-phase cells, wild-type IEC-6, EGFR-k/d IEC-6 or GSK3β-k/d IEC-6 cells were plated on cover slips in 12-well cell culture dishes overnight, treatment groups as indicated in Results along with BrdU labeling reagent (10uL/ml media, Invitrogen) for 6 hrs. To detect BrdU incorporation, cells were fixed with 4% PFA on ice for 10 min., processed for antigen retrieval with 2M Hydrochloric acid for 30 min, blocked with 5% donkey serum in 0.5% bovine serum albumin (BSA), incubated overnight at 4 degrees Celsius with rat-anti-BrdU antibodies (Novus Biosciences) and detected with cy3-labelled secondary anti-rat antibodies using a confocal microscope (Zeiss).
6
TUNEL Assay for Apoptosis Quantification
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Cells were grown on glass coverslips. After treatment with WP1066 for 48 h, TUNEL staining was performed using Click-iT TUNEL Alexa Fluor 594, as recommended by the supplier (Invitrogen). Cells were counterstained with Hoechst dye for the nuclei and fluorescent images were obtained using a confocal microscope (Zeiss). The results are from three independent experiments. The apoptotic cell rate was determined according to the formula: (number of apoptotic cell with red staining/total number of cells with 4′-6-diamidino-2-phenylindole staining) × 100%.
7
Phagocytosis Comparison in RPE Cells
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To evaluate the functionality of the RPE, the ability of the phagocytosis was compared between RPE cells cultured on BM-ECM and laminin-coated Transwells. Each sample was incubated with 2 μm of fluorescence-labeled polystyrene bead (Invitrogen) at a concentration of 1 × 107/mL for 16 h. After incubation, RPE cells were washed with PBS three times to remove undigested beads. The cells were fixed with 4% paraformaldehyde solution for 20 min and permeabilized with 0.03% Triton-X for 7 min at room temperature. After permeabilization, the samples were incubated with DAPI and TRITC phalloidin (Sigma-Aldrich). Imaging was carried out using a confocal microscope (Zeiss).
8
Immunofluorescent Analysis of Renal α-SMA
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Kidney were fixed in 4% PFA for 1 h at 4°C, cryopreserved in 20% sucrose, and frozen in liquid nitrogen. Five micrometers sections were stained for α-SMA using a mouse fluorescein isothiocyanate-conjugated antibody (Sigma). Images were made using a confocal microscope (Carl Zeiss, Sliedrecht, Netherlands).
9
Fluorescent In Situ Hybridization Protocol
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The fluorescent in situ hybridization kit was purchased from Ribo Bio (Guangzhou, China) and the experiment is performed according to the manufacturer’s instruction and previously described [10 (link)], then visualized by a confocal microscope (Zeiss, Oberkochen, German). The CY3 labeled 18S probes were provided by the Ribo Bio (Guangzhou, China) and the LBCS probe was synthesized by Sangon (Shanghai, China). The sequences of LBCS and U6 probes were listed in Additional file 3 : Table S3.
10
Exosome Internalization Protocols
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Macrophages were cultured in medium containing 10% exosome-free FBS for 48 h. The medium was collected for extraction of exosomes with an exoEasy Maxi Kit (QIAGEN, Germany) following the manufacturer’s instruction.
To determine exosome internalization, the exosomes were labeled with PKH67 (Sigma) according to the manufacturer’s protocol. Subsequently, the labeled exosomes were used to incubate with cells in each experiment, and observed using a confocal microscope (Carl Zeiss, Germany).
To determine exosome internalization, the exosomes were labeled with PKH67 (Sigma) according to the manufacturer’s protocol. Subsequently, the labeled exosomes were used to incubate with cells in each experiment, and observed using a confocal microscope (Carl Zeiss, Germany).
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