Reagents and material for cell culture
Dulbecco’s Modified Eagle Medium (DMEM), Gibco, 11965-084; Fetal bovine serum (FBS), HyClone, SH30084.03; L-Glutamine, Gibco, A29168-01; Penicillin/Streptomycin, Gibco, 15140-122; 10X PBS, pH 7.4, Lonza, 17-517Q, 10-fold dilution before use; 0.05% Trypsin-EDTA, Gibco, 25300-054; poly-L-lysine, Sigma-Aldrich, P26360, 1 mg/ml; No. 1 coverslip, 15-mm in diameter, Fisher, 1942–10015.
Cell preparation
The COS-7 cell line (ATCC, CRL-1651), an African green monkey kidney fibroblast-like cell line, was maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 2 mM glutamine in a 5% CO2 incubator at 37°C. To prepare cell samples for expansion microscopy, COS-7 cells that grew to 100% confluency in a 10-cm culture dish were harvested and resuspended in 10 ml DMEM. The cell suspension (0.4 ml) was added into one well of a 12-well plate containing DMEM growth medium (1 ml/well) with a poly-L-lysine-coated coverslip. The cells were then placed back in a 5% CO2 incubator at 37°C. One day later, cells were ready for immunostaining. Hippocampal and cortical mixed cultures were kindly provided by Dr. Yi-Ping Hsueh’s laboratory.
Reagents for immunofluorescence staining
Paraformaldehyde (PFA), Merck, 1.04005.1000; Triton X-100, Sigma-Aldrich, T8787; Tris, VRW, 0826; Blocking reagent for immunostaining, Tyramide Signal Amplification kit, PerkinElmer, 581428; Cytochrome c oxidase subunit 5B (COX5B) rabbit polyclonal antibody, Proteintech, 11418-2-AP, 3–6 µg/ml; Protein disulfide isomerase (PDI) rabbit polyclonal antibody, Proteintech, 11245-1-AP, 1–2 µg/ml; Beta-tubulin mouse monoclonal antibody, Sigma-Aldrich, T4026, 6 µg/ml; Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, secondary antibody, Jackson ImmunoResearch Laboratory, 711-035-152, 1:500 dilution; Alexa fluor-488-conjugated anti-mouse IgG, secondary antibody, ThermoFisher, A28175, 1:500 dilution; Alexa fluor-555-conjugated anti-rabbit IgG, secondary antibody, ThermoFisher, A32732, 1:500 dilution; Alexa fluor-555-conjugated tyramide, Invitrogen, B40955, 1:100 dilution; Amplification diluent, PerkinElmer, FP1050; PicoGreen, ThermoFisher, P11495, 1:1000 dilution.
Immunostaining with tyramide signal amplification
COS-7 cells that grew on coverslips were first washed with PBS three times and fixed with 4% PFA in PBS for 15 min at room temperature for regular staining. Alternatively, cells were washed three times with PBS pre-warmed to 37°C and then fixed with 4% PFA in PBS pre-warmed to 37°C for 30 min at 37°C to observe mitochondria in cells. After washing twice with Tris-buffered saline (TBS), TBST (TBS + 0.3% Tritin-X-100) was added, before incubating for 10 min at room temperature for permeabilization. After rinsing once with TBST, 0.5% Blocking solution was added for 30 min at room temperature. Cells were then incubated with the primary antibodies diluted in blocking solution for 2 h at room temperature or overnight at 4°C. After washing three times with TBST, secondary antibodies were added and incubated for 1.5 h at room temperature. After rinsing three times with TBST to remove unbound antibodies, Alexa fluor-555-conjugated tyramide was diluted 100-fold with amplification diluent and applied to cells for 10–20 min at room temperature. The cells were then rinsed three times with TBST to stop the reaction. Before crosslinking, immunofluorescence signals were checked under an inverted fluorescence microscope to confirm successful staining.
Reagents for lipid staining
Paraformaldehyde (PFA), Merck, 1.04005.1000; 1x PBS; N-(Biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Biotin-DHPE), Biotium, 60022; VECTASTAIN ABC-HRP Kit, peroxidase (standard), Vector Laboratory, PK-4000; Alexa fluor-555-conjugated tyramide, Invitrogen, B40955, 1:100 dilution; Amplification diluent, PerkinElmer, FP1050; PicoGreen, ThermoFisher, P11495, 1:1000 dilution.
Lipid staining using tyramide signal amplification
Biotin-DHPE was dissolved in chloroform at a concentration of 10 mg/ml. After washing three times with PBS, COS-7 cells or cultured neurons were fixed with 4% PFA in PBS for 15 min at room temperature. To observe mitochondria, the PBS and 4% PFA were pre-warmed to 37°C directly before use. After washing three times with TBS, fixed cells were transferred to 70% or 50% ethanol containing 0.1 mg/ml biotin-DHPE and incubated for 2.5 h or overnight at room temperature. An equal amount of reagents A and B of an ABC-HRP Kit was mixed in TBS at a dilution of 1:50 for at least 30 min. Meanwhile, unbound biotin-DHPE was removed by extensively washing at room temperature. The premixed ABC solution was then added to biotin-DHPE-labeled cells for 1 h. After washing, Alexa fluor-555-conjugated tyramide was diluted 1:100 in amplification diluent and applied to cells for 15 min at room temperature. After extensive washing, the stained cells were then ready for anchoring to a hydrogel.
Reagents for anchoring and making the acrylamide hydrogel
6-((acryloyl)amino)hexanoic acid (Acryloyl-X, SE), ThermoFisher, A-20770; Dimethyl sulfoxide (DMSO), Thermo Fisher, D12345; Sodium acrylate (SA), Sigma-Aldrich, 408220; Acrylamide (AA), Sigma-Aldrich, A9099; N, N’-methylenebisacrylamide (MBAA), ThermoFisher, 1551624; Ammonium persulfate (APS), Sigma-Aldrich, A9164; N,N,N,,N,-Tetramethylethylenediamine (TEMED), Sigma-Aldrich, T7024; 4-Hydroxy-TEMPO, ALDRICH, 176141; Sodium Chloride, J.T.Baker, 3624-69.
Anchoring
Before gelation, the stained cells were incubated with acryloyl-X, SE to crosslink protein molecules with the acrylamide gel. Acryloyl-X, SE was first dissolved in DMSO at a concentration of 10 mg/ml and then diluted to 0.1 mg/ml in PBS right before adding it to the stained cells. The samples were incubated overnight at 4°C for reaction. Directly before gelation, the samples were washed three times with PBS at room temperature to remove excess Acryloyl-X, SE.
Gelation
To make sufficient acrylamide solution for six gel discs of 0.5 mm thickness and 15 mm diameter, 564 µl acrylamide monomer solution (containing 8.6% sodium acrylate, 2.5% acrylamide, 0.15% N,N’-methylenebisacrylamide and 11.7% sodium chloride in PBS) was mixed with 12 µl 10% TEMED, 6 µl 1% TEMPO, 6 µl water and 12 µl 10% APS. After extensively vortexing, 88 µl of the mixture was immediately added onto parafilm, which was placed in a 3.5-cm dish. After removing the extra PBS, a coverslip was placed on top of the acrylamide mixture with the side hosting cells facing down. Gelation was achieved at 37°C for 2 h.
Enzyme digestion and expansion of the hydrogel
Approximately a quarter of the hydrogel disc was cut with a scalpel and measured using graph paper. The gel was then gently picked up with a flat brush and placed in a solution containing a protease for hydrolysis. We tested two proteases, i.e., proteinase K and trypsin, incubated either for 2 h or overnight at room temperature. The concentrations of these two enzymes, the composition of the digestion buffer, and their cleavage sites are listed in Table 1. The digested hydrogel was completely immersed in double-distilled water in a 10-cm petridish. After at least 30 min, the double-distilled water was replaced with fresh water and this process was repeated at least three times. The size of the swollen hydrogel was again measured using graph paper.
Table 1
Comparison of proteinase K and trypsin digestion.
| proteinase K | Trypsin (0.05%, Gibco 25300-054), low-salt | Trypsin (0.05%, Gibco 25300-054), high-salt |
Working concentration of the protease | 8 units/ml | 0.015% | 0.015% |
Ingredients of the digestion buffer | Tris, pH 8.0, 50 mM; EDTA, 1 mM; Triton-X-100, 0.5%; *Guanidine HCl, 0.8 M | Tris, pH 8.0, 35 mM; EDTA, 0.7 mM; Triton-X-100, 0.35% | Tris, pH 8.0, 35 mM; EDTA, 0.7 mM; Triton-X-100, 0.35% NaCl, 0.5 M |
Cleavage sites of the proteases | Aliphatic or aromatic amino acids, including leucine (7.6%), alanine (7.4%), valine (6.8%), isoleucine (3.8%), glycine (7.4%), phenylalanine (4%), proline (5%), tryptophan (1.3%) or tyrosine (3.3%)** | Lysine (7.2%) or arginine (4.2%)** |
* Guanidine HCl is a chaotropic agent, which can destroy intramolecular and intermolecular hydrogen bonds, thereby unraveling the three-dimensional structure of proteins and nucleic acids, which increases the hydrolytic efficiency of proteases. This chemical was only used in the proteinase K-based hydrolysis reaction, and not in the trypsin reaction.
** The percentages shown in parentheses after the amino acids represent the average frequency of use of each amino acid in vertebrates 40, thereby reflecting the number of times that the protease cleaves a protein molecule with a length of 100 amino acids under ideal conditions.
Poststaining with PicoGreen DNA dye
The expanded hydrogel was placed in water containing the DNA dye PicoGreen (1:1000) for at least 2 h at room temperature. To completely penetrate into nuclei, PicoGreen staining longer than overnight at 4°C was required.
Imaging using a LSM700 confocal laser scanner and image processing
An appropriate size (~ 0.5 x 1 cm) of the expanded hydrogel containing COS-7 cells was placed on a 24x50 mm coverslip with the side hosting cells facing down. The excess water was carefully removed. The expanded samples were then observed under an inverted confocal microscope LSM700 (Zeiss) with a Plan Apo 20X/NA = 0.8 DICII objective lens at room temperature. Non-expanded samples were directly mounted and observed using the same 20X objective lens under the same microscope for comparison with expanded samples. Fluorescence images were captured with Zen software 2009 (https://www.zeiss.com/microscopy/en/products/software.html) from the Zeiss Corporation. Single focal plane images were collected and then analyzed using ImageJ/Fiji software (V 2.1.0/1.53c, https://imagej.nih.gov/ij/index.html). The images were then converted into a format recognizable by general image software and assembled using Adobe Photoshop 2020.
Imaging using a confocal laser scanner LSM980 system with Airyscan2 and image processing using Labkit and Imaris
For cultured neurons, the expanded gel was placed in a water-filled mold on a 0.17-mm-thick coverslip. Images were recorded using a LSM980 laser scanning confocal microscope equipped with multiplex mode SR-4Y (2X sampling) and an Airyscan 2 detector at room temperature. A LD LCI Plan-Apochromate 40X/1.2 Imm Korr DIC M27 objective was adopted for image acquisition. We applied 488 nm and 561 nm laser excitations for DNA and lipid signals, respectively. The 1844x1844 pixel images in the Z series were acquired at 0.2 µm intervals. The Z-series images were further processed to conduct 3D surface construction using Imaris 9.9.1 software with the machine learning Fiji-Labkit plugin for segmentation. The shortest distance and smooth options were chosen as Imaris parameter settings. The channel of interest was then directed to Labkit. In Labkit, the pencil tool was used to draw on images and identify "foreground" and "background" regions. This segmentation was sent back to Imaris to create the surface. In neurons, mitochondrion-like surfaces were defined by tubular lipid signals with a minimal object-object distance to DNA signals. The images were then converted into a format recognizable by general image software and assembled using Adobe Photoshop 2020.
To calculate the expansion ratios, we first identified and paired the same cell that appeared in the pre-and post-expansion images. The pre-expansion images were manually adjusted to match the post-expansion images, necessitating translation, rotation, and scaling. This series of operations generated a coarse expansion ratio between the pre-and post-images. To refine the expansion ratio, we used the "register image" function in Amira software from Thermo Scientific to examine the images further. Expansion ratios were calculated based on 15 cells. The average and median values are presented as horizontal bars and white circles, respectively, in Fig. 4B, 4C. The boundary of the distribution in the violin plots denotes the lower and upper bound of the dataset.