Cells in Figures 1 and 3 and Supplemental Figures S1 and S2 were cultured in yeast extract/peptone/dextrose (YPD) medium at room temperature, fixed with 4% paraformaldehyde (Boston Bio Products), and washed at least three times in 1× phosphate-buffered saline (PBS) before imaging. For live-cell imaging in Figures 2 and 5 , cells were cultured in synthetic complete plus 2% dextrose medium at room temperature. For fluorescence microscopy of Figure 4 , cells were cultured in YP plus 2% raffinose at room temperature and arrested in 10 μg/ml nocodazole for 4 h, and 2% galactose was subsequently added for an additional 2 h. Cells were fixed in 4% paraformaldehyde (Boston Bio Products) and washed at least three times in 1× PBS before imaging. Some cells of the cdh1∆ strains appeared abnormally large and were excluded from the analysis. Cells in Figure 6A were cultured in YP plus 2% raffinose overnight at 30°C overnight and released at low density into YP plus 2% galactose medium at 30°C the next day for 6 h. For the fluorescence microscopy of Figure 6B and Supplemental Figure S3, cells were cultured in YP plus lactate medium overnight at 30°C and released at low density into YP + 2% galactose medium at 30° the next day for 6 h. Cells were fixed in 4% paraformaldehyde (Boston Bio Products) and washed at least three times in 1× PBS before imaging.
Paraformaldehyde (pfa)
Manufactured by Boston BioProducts
Sourced in United States
Paraformaldehyde is a solid polymer of formaldehyde, commonly used as a fixative in various laboratory applications. It is a white, crystalline powder that slowly releases formaldehyde gas. Paraformaldehyde is a versatile material that can be used to preserve biological samples and tissues for microscopic analysis.
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Lab products found in correlation
Heracell 150i, by Thermo Fisher Scientific (6 mentions)
Lsm 710 confocal microscope, by Zeiss (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (3 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (3 mentions)
Lsr 2, by BD (3 mentions)
Emd millipore millicell ez slides, by Merck Group (2 mentions)
Apotome 2 microscope, by Zeiss (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Antibody against nf κb2 p100 52, by Cell Signaling Technology (2 mentions)
Cold methanol, by Thermo Fisher Scientific (2 mentions)
Rat anti human her2, by Agilent Technologies (2 mentions)
Mouse anti human cd3, by Agilent Technologies (2 mentions)
Rat anti human granzyme b, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Apc conjugated mouse anti his antibody, by BioLegend (2 mentions)
Avicel, by FMC Biopolymer (2 mentions)
Nahco3, by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Boston BioProducts (2 mentions)
36 protocols using paraformaldehyde (pfa)
1
Fluorescence Microscopy of Yeast Cell Cultures
2
VEGF-Induced Endothelial Cell Imaging
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HUVEC, HPAEC, or HMVEC-L were seeded at 4 × 104 cells per well onto EMD Millipore Millicell EZ Slides (PEZGS0416; Sigma Millipore), grown in complete medium for 48 hours, and subsequently serum starved for 16 hours. After receiving inhibitors and/or human recombinant VEGF-165 stimulation, cells were fixed in 4% paraformaldehyde (Boston Bioproducts), permeabilized in cold methanol (Fisher), and immunofluorescence staining was performed. Nuclei were stained using 4’, 6-diamidino-2-phenylindole (DAPI; Cell Signaling; Catalog No. 8961S). All images were captured on a Zeiss Apotome 2 microscope by using 20×, 0.8 NA Plan Apochromat objective magnifying 2X to 4X and processed using ImageJ software (Version 1.8.0_112; https://imagej.nih.gov/ij ).
3
Tumor Tissue Fixation and Immunohistochemistry
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Tumor tissue was harvested at the experimental endpoint and fixed in 3.7% paraformaldehyde (Boston BioProducts) or 10% neutral buffered formalin (Sigma). Tissue was processed for paraffin embedding, sectioning and H&E staining by the Harvard Rodent Histopathology core. Unstained sections were analyzed by immunohistochemical analysis according to standard protocol. For expanded details on immunohistochemistry methods and antibodies, please refer to the Supplementary Methods .
4
Confocal Imaging and Tracing of Interneurons
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Slices were fixed in 4% paraformaldehyde (Boston BioProducts) and incubated with streptavidin Alexa Fluor 633 (Life Technologies, Invitrogen) in phosphate buffered saline (PBS) with Triton-X 100 as previously described (Bell et al., 2011 (link)). Processed slices were then reconstructed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Alexa Fluor 633 was excited with the 633 nm line of a HeNe 5 mW laser and cells were visualized using a 20× dry lens (0.8 N.A., voxel dimensions 0.2 × 0.2 × 1.1 µm). The imaged interneurons were traced using the Autoneuron module within the Neurolucida program (MBP, Burlington, VT). For amplification of YFP-labeled interneurons, 1:200 dilution of rabbit anti-GFP conjugated to Alexa Fluor 488 (Life Technologies, Invitrogen) in goat blocking buffer (10% normal serum, 2% bovine serum albumin, 0.4% Triton-X 100 in 0.1 M phosphate buffer) was added to fixed and washed slices for overnight incubation. Before and after primary and secondary antibody incubations, slices were washed in PBS. Slices were mounted in either Prolong Gold® (Life Technologies, Invitrogen) or VECTASHIELD® hard mount (Vector Laboratories).
5
VEGF-induced Endothelial Cell Imaging
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HUVECs, HPAECs or HMVEC-Ls were seeded at 4×104 cells per well onto EMD Millipore Millicell EZ Slides (PEZGS0416, Sigma Millipore), grown in complete medium for 48 h and subsequently serum starved for 16 h. After receiving inhibitors and/or human recombinant VEGF-165 stimulation, cells were fixed in 4% paraformaldehyde (Boston Bioproducts), permeabilized in cold methanol (Thermo Fisher Scientific), and immunofluorescence was performed. Nuclei were stained using 4′, 6-diamidino-2-phenylindole (DAPI; 8961S, Cell Signaling Technology). All images were captured on a Zeiss Apotome 2 microscope using a 20×/0.8 NA Plan Apochromat objective magnifying 2× to 4× and processed using ImageJ software.
6
Comparative Morphology of Deep-Sea Shrimps
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The three species of deep‐sea shrimps used in this study, Parasergestes armatus, Allosergestes sargassi, and Deosergestes henseni, represent the three major organs of Pesta morphologies (i.e., bilobed, trilobed, and fringed, respectively; Figure 1b–d ). The animals for this study were obtained during two deep‐sea research expeditions, the first occurring in the Straits of Florida on May 4–8, 2019, on the R/V Weatherbird and the second occurring in the Northern Gulf of Mexico on June 9–22, 2019, as part of a NOAA Ocean Research Exploration expedition on the R/V Point Sur. Animals were captured by 1 or 9‐m2 tucker trawl over sampling events that occurred both day and night over a total range of depths from 150–1,500 m. Animals captured on the first expedition were immediately placed in 4% paraformaldehyde in 0.1 M phosphate‐buffered saline (PBS; Boston BioProducts, Ashland, MA, USA) for a 48‐hr fixation step prior to transfer into 0.1 M PBS for subsequent analyses. Those from the second expedition were examined immediately, allowing us to obtain morphological data from fresh animals prior to fixation.
7
Quantifying CD155 Expression in Tumor Cells
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Immunofluorescence was used to analysis the expression of CD155 in tumor cells (Hela and Skov3). The slides with cells were fixed with 4% paraformaldehyde (Boston Bioproducts) for 15 min. Normal goat serum was added to the slides were sealed at room temperature for 30 min. Each slide was dropped with enough Human CD155/PVR Antibody (R&D, Minneapolis, MN, USA) and placed in a wet box for incubation at 4°C overnight. After incubation, sample were washed and incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (Abcam, Grand Island, NY) for 60 min at room temperature. The samples were stained DAPI (R&D, Minneapolis, MN, USA) and incubated for 5 min. Then PBST was washed for 5 min, 4 times to remove the excess DAPI. Dry the liquid on the slipper with absorbent paper, seal the slipper with sealing liquid containing anti-fluorescence quench agent, and observe and collect the image under Laser scanning confocal microscope (LSCM).
8
Tumor Tissue Histopathological Analysis
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Tumor tissue was fixed for up to 3 days in 4% paraformaldehyde (Boston BioProducts) and stored in 70% ethanol until further processing. Histology was performed by the Pathology Core at City of Hope. Briefly, paraffin-embedded sections (10-µm) were stained with hematoxylin & eosin (H&E, Sigma-Aldrich), mouse anti-human CD3 (DAKO), mouse anti-human PSCA (Abcam), rat anti-human HER2 (DAKO), and rat anti-human Granzyme-B (eBioscience). Images were obtained using the Nanozoomer 2.0HT digital slide scanner and the associated NDP.view2 software (Hamamatzu).
9
Quantifying Cerebral Microhemorrhage Characteristics
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Right hemispheres were fixed in 4% paraformaldehyde (Boston BioProducts, Ashland, MA) at 4 °C, examined for surface CMH, and sectioned into 40-μm coronal sections using a vibratome (Technical Products International, Inc., St. Louis, MO). Every fourth, fifth, sixth, and seventh section was collected. Every sixth section was stained with H&E by Research Services Core offered by the Department of Pathology and Laboratory Medicine at UCI Medical Center to detect fresh (acute) CMH. Every seventh section was stained with Prussian blue (PB) to detect hemosiderin (a marker of sub-acute CMH) [16 (link)]. Briefly, sections were stained using 5% potassium hexacyanoferrate trihydrate (Sigma, St. Louis, MO) and 5% hydrochloric acid (Sigma, St. Louis, MO) for 30 min, rinsed in water and counterstained with Nuclear Fast Red (Sigma, St. Louis, MO), dehydrated, and cover slipped. Remaining sections were used for immunohistochemistry. CMH were counted at a × 20 magnification by a blinded observer, and digitized images were used to determine CMH size (μm2) and positive area (expressed as a percent of total area analyzed), by an observer blinded to the experiment using RGB CMH analyzer program and NIH Image J software 1.62, respectively [16 (link)].
10
Osteoblast Attachment on Magnesium Alloys
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Mouse osteoblasts (MC 3T3, ATCC, USA) was expanded in Minimum Essential Medium α (MEM, Life Technologies, USA) supplemented with 10% fetal bovine serum (Sciencell, USA), 100 U/ml penicillin (Sciencell, USA) and 100 µg/ml streptomycin (Sciencell, USA) in humidified incubator (Heracell 150 I, Thermo, USA) with 5% CO2 as previously described [35] (link). 50 µl 200 µg/ml collagen solution (pH of 7) was allowed to self-assemble on Mg and AZ31 with RS, SR, and SS for 2 h in a 24-well culture plate (BD Bioscience, USA). Then these materials were gently rinsed by DPBS for 3 times. 50 µl cell solution with density of 10,000 cell/ml was dipped onto the surface of collagen treated materials. Cells were allowed to attach for 30 min and then samples were washed gently with DPBS for 3 times. After 4 hours, cells were fixed with 4% paraformaldehyde (Boston Bioproducts, USA) followed by ethanol gradient dehydration for 10 minutes. Samples were coated (E5400, Polaron Instruments, USA) with gold nanoparticles for 2 min, and imaged by SEM.
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