After paraffin removal and rehydration, the slides were heated in 1× citrate buffer (ThermoFisher) and exposed to 3% H2O2 (ThermoFisher) at room temperature and then the blocking solution. After washing with PBS plus 0.1% Tween 20 (J.T. Baker), the tissue was incubated with primary antibodies at 4°C overnight. The primary antibodies of AQP2, NHE3, NKCC2, NCC, p-NCC, ENaCβ, Maxi-K, ROMK, and pendrin were used. The tissues were exposed to species-specific secondary antibodies conjugated to Alexa Fluor fluorophores (ThermoFisher). Immunofluorescence images were obtained by Zeiss LSM880 confocal microscope.
Alexa fluor fluorophores
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Alexa Fluor fluorophores are a series of synthetic dyes developed by Thermo Fisher Scientific. These fluorophores are designed to emit fluorescence when excited by specific wavelengths of light, enabling their use in various biological and analytical applications, such as fluorescence microscopy, flow cytometry, and immunoassays. Alexa Fluor fluorophores offer a range of emission wavelengths, providing researchers with options to select the appropriate dye for their specific needs.
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Lab products found in correlation
Donkey serum, by Jackson ImmunoResearch (2 mentions)
Delta vision imaging system, by Cytiva (2 mentions)
Dapi fluoromount g, by Southern Biotech (2 mentions)
Citrate buffer, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Anti rab11a, by Santa Cruz Biotechnology (1 mentions)
Anti eea1, by Santa Cruz Biotechnology (1 mentions)
Anti gamma tubulin, by Merck Group (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Retriever 2100, by Proteogenix (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Histogel, by Thermo Fisher Scientific (1 mentions)
Histoclear, by Thermo Fisher Scientific (1 mentions)
A1rsi confocal microscope, by Nikon (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Dmi4000 microscope, by Leica (1 mentions)
A1rsi confocal microscope, by Leica (1 mentions)
Rabbit anti g3bp1, by Thermo Fisher Scientific (1 mentions)
12 protocols using alexa fluor fluorophores
1
Immunofluorescence Localization of Renal Transporters
2
Immunophenotyping of Immune Cells
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Sections from each case were also immunostained to detect CD4+, CD8+ and CD20+ cells using validated antibodies, as described previously [8 (link), 9 (link)]. Primary antibodies are listed in ESM Table 6 and the immunohistochemical analysis was performed using Agilent/DAKO Envision reagents (Agilent Technologies, Cheadle, Cheshire, UK) or Alexafluor fluorophores (ThermoFisher, Loughborough, UK) with or without the Tyramide SuperBoost (ThermoFisher) protocol. Immunophenotyping was achieved as described previously [8 (link)].
3
Immunofluorescence Labeling of Organelles
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Immunofluorescence experiments utilized 4% bovine serum albumin (BSA, Sigma–Aldrich, A7030) in PBS as blocking reagent and antibody diluent. Where necessary, fixed cells were permeabilized with 0.1% Triton-X 100 in PBS prior to blocking. Dextran labeled cells were not permeabilized, but utilized 0.05M glycine in the blocking media. Actin filaments were labeled with Alexa-647 conjugated Phalloidin dyes (ThermoFisher Scientific, A22287) per the manufacturer protocol. Primary antibodies used were anti-Alpha tubulin (1:500, Abcam ab7291), anti-ARL13B (1:250, ProteinTech 17711-1-AP), anti-gamma tubulin (1:250, Sigma–Aldrich T5326), anti-EEA1 (1:200, SantaCruz Biotechnology sc-6414), anti-LAMP3 (1:200, ThermoFisher Scientific, MA1-35272), anti-Rab9 (1:500, ThermoFisher Scientific, MA3-067), anti-Rab4 (1:250, ThermoFisher Scientific, PA3-912), anti-Rab11a (1:250, SantaCruz Biotechnology sc-166912), and anti-Rab7 (1:100, ThermoFisher Scientific, PA5-52369). Secondary antibodies used were donkey anti-rabbit, anti-mouse or anti-goat and conjugated to AlexaFluor fluorophores (ThermoFisher Scientific, 1:1000 dilution). Primary and secondary antibodies were diluted in 4% BSA in PBS. DNA counterstaining utilized Hoechst33342 dye (ThermoFisher Scientific, H1399), diluted 1:5000 in PBS.
4
Immunostaining of Pancreatic Islets
5
Immunostaining of Pancreatic Cell Markers
6
Visualizing Stress Granule Formation
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Cells expressing SOD1(A4V)‐GFP were exposed to heat stress for 2 h and fixed with 4% PFA (diluted in PBS) for 10 min. This was followed by washing with PBS and then permeabilization with 0.2% Triton X‐100 (diluted in PBS) for 10 min. The cells were then washed with 2× SSC. Hybridization was then performed by incubating the cells for 2 h at 37°C in 4× SSC containing 10% formamide, 5% dextran sulfate, 1% BSA, 0.5 mM EDTA, and 100 nM biotinylated oligo‐dT probe (23‐mer). This was followed with washing in 2× SSC (3 × 10 min) and blocking in 3% BSA (diluted in 4× SSC) for 1 h at room temperature. The cells were then incubated with primary antibodies (goat anti‐biotin, Sigma‐Aldrich, B3640; rabbit anti‐G3BP1, Thermo Fisher Scientific, PA5‐29455) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC and incubated with secondary antibodies conjugated with Alexa Fluor fluorophores (Invitrogen) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC, then washed in 2× SSC, and finally mounted in DAPI‐Fluoromount G (SouthernBiotech). The cells were then imaged using the DeltaVision imaging system (Applied Precision) as described above.
7
Comprehensive Immunolabeling of Neural Cell Types
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Primary antibodies included those to GFP (rabbit, 1:1000 dilution, MBL, 598; chicken, 1:1000, Abcam, ab13970), GFAP (rabbit, 1:1000, Dako, Z0334; mouse, 1:500, Millipore, MAB360), NeuN (mouse, 1:200, Millipore, MAB377), S100β (mouse, 1:500, Sigma, SH-B4; rabbit, 1:1000, Dako, Z0311), S100 (mouse, 1:500, Abcam, ab4066), Sox10 (goat, 1:200, Santa Cruz Biotechnology, sc17342), Cux1 (rabbit, 1:1000, Santa Cruz Biotechnology, sc13024), CTIP2 (rat, 1:2000, Abcam, ab18465), Zbtb20 (mouse, 1:200, Abcam, ab48889), HMGN1 (rabbit, 1:1000, Abcam, ab5212), HMGN2 (rabbit, 1:500, LifeSpan Biosciences, LS-C118756), HMGN3 (rabbit, 1:100, Santa Cruz Biotechnology, sc138955), Id1 (rabbit, 1:200, Biocheck, BCH-1/37-2), and Lef1 (rabbit, 1:1000, Cell Signaling, #22). Secondary antibodies included those conjugated with Alexa Fluor fluorophores to mouse, rabbit, chicken, rat, or goat immunoglobulin G (1:500, Invitrogen).
8
Investigating Tau and ER Stress Markers
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The primary antibodies used in this study were anti-actin (Sigma, AC-40), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2896), anti-p-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 9722), anti-tau (Dako, A0024), anti-GFAP (Dako, Z0334), anti-NeuN (Cell Signaling Technology, 12943), anti-GAPDH (Abcam, ab8245), and MC-1 and PHF-1 (gifts from Prof. Peter Davies) (17 (link)). Secondary antibodies were conjugated to Alexa Fluor fluorophores (Invitrogen) or IRDyes (LI-COR Biosciences). The plasmids pRK5-EGFP-TauP301L and pRK5-EGFP-Tau were gifts from Karen Ashe (Addgene plasmids 46908 and 46904, respectively) (32 (link)), and GFP in the constructs was exchanged for RFP.
Primers were obtained from Eurofins Genomics: BiP, 5-ATTGGAGGTGGGCAAACCAA-3 (forward) and 5-TCGCTGGGCATCATTGAAGT-3 (reverse); CHOP, 5-TCCCCAGGAAACGAAGAGGAAG-3 (forward) and 5-TCATGCGTTGCTTCCCAGGC-3 (reverse); XBP-1, 5-GGAGTGGAGTAAGGCTGGTG-3 (forward) and 5-GTCCAGAATGCCCAAAAGGATA-3 (reverse). Primers for the reference gene eIF4a were purchased from Primer Design.
Primers were obtained from Eurofins Genomics: BiP, 5-ATTGGAGGTGGGCAAACCAA-3 (forward) and 5-TCGCTGGGCATCATTGAAGT-3 (reverse); CHOP, 5-TCCCCAGGAAACGAAGAGGAAG-3 (forward) and 5-TCATGCGTTGCTTCCCAGGC-3 (reverse); XBP-1, 5-GGAGTGGAGTAAGGCTGGTG-3 (forward) and 5-GTCCAGAATGCCCAAAAGGATA-3 (reverse). Primers for the reference gene eIF4a were purchased from Primer Design.
9
Immunofluorescence Analysis of Cellular Proteins
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HeLa cells grown on coverslips were fixed using 4% PFA in PBS. Permeabilization was performed using 0.2% Triton X‐100 in PBS for 10 min. 3% BSA in PBS was used for blocking. All antibodies were diluted in 1% BSA in PBS. The following antibodies were used: goat anti‐eIF3η (N‐20, Santa Cruz Biotechnology), mouse anti‐polyubiquitin (FK1, Enzo Life Sciences), mouse anti‐HSP27 (ADI‐SPA‐800, Enzo Life Sciences), mouse anti‐HSP70 (ADI‐SPA‐810, Enzo Life Sciences), mouse anti‐VCP (MA3‐004, Thermo Fisher Scientific), mouse anti‐p62/SQSTM1 (D‐3, Santa Cruz Biotechnology), mouse anti‐TARDBP (41‐7.1, Santa Cruz Biotechnology), and mouse anti‐vimentin (V9, Santa Cruz Biotechnology). Secondary antibodies conjugated with Alexa Fluor fluorophores (Invitrogen) were then applied, followed by washing in PBS. Coverslips were mounted on microscope slides in DAPI‐Fluoromount G (SouthernBiotech). The cells were then imaged using the DeltaVision imaging system (Applied Precision) or the Scan^R imaging platform (Olympus), as described above.
10
Antibody and Plasmid Characterization
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Primary antibodies used in this study were anti-FLAG M2 (Merck, F1804), anti-α-Actin (Sigma, AC-40), anti-Task3 (Almone, APC-044), anti-DHX36 (Proteintech, 13159), anti-α-Tubulin (Proteintech, 66031), anti-Lamin A+C (Cell Signalling Technology, 2032), BG4 (Absolute, Ab00174-24.1). Secondary antibodies used were conjugated to Alexa Fluor® fluorophores (Invitrogen) or IRDyes (LI-COR Biosciences). The plasmids used were cloned from human brain cDNA (Agilent 540005-41) into C-terminal pcDNA3.1-3F/eGFP vectors as previously described (35 (link)), and mutants created through site-directed mutagenesis (Promega). Primers were obtained from Sigma, with sequences outlined in Supplementary Figure S6 . Stellaris® RNA FISH probes were obtained from LGC Biosearch Tech. Anti-Task3 mouse RNA FISH probes were designed using the Stellaris® online web tool (see data availability) and conjugated to Quasar® 570 nm fluorophores. Anti-eGFP RNA FISH probes were pre-designed from Stellaris® and conjugated to Cal Fluor® Red 590 nm fluorophores.
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