4-benzaldehydesulfonic acid

Following the RNAscope protocol, 28-hpf embryos were embedded in 4% LMP and sectioned into 100-μm slices using a vibratome (Leica, Wetzlar, Germany VT1000E). Sections were permeabilized (0.1% Tween-20 and 0.3% Triton-X-100 in PBS) for 1 hour at RT and incubated in blocking buffer (0.3% Triton X-100 and 4% BSA in PBS) O/N at 4°C. Incubation with a mouse monoclonal primary antibody targeting E-cadherin (BD, San Jose, CA, USA Biosciences, 610181) in blocking buffer (1:100) was performed O/N at 4°C followed by three washes for 5 min in 0.3% Triton X-100 in PBS. The secondary antibody (rabbit Alexa 568 anti-mouse IgG, Invitrogen) was applied at 1:1000 dilution in blocking buffer and incubated O/N at 4°C in the dark. The sections were then washed three times, mounted on slides using fluorescence mounting medium (Dako, Hamburg, Germany) and imaged.

GLA activities were determined in Fabry mouse tissues using previously described methods (Desnick et al., 1973 (link)). In brief, tissue samples were homogenized in chilled reporter lysis buffer (Promega) and protease inhibitor (Pierce) was added to the lysates. Protein concentrations were determined using the Bio-Rad Colorimetric Protein Assay Kit. 10 μL of tissue lysate was added to an equal volume of 10 mM 4-methylumbelliferyl-α-D-galactopyranoside (Sigma-Aldrich), dissolved in assay buffer (0.2 M citrate, 0.4 M phosphate buffer, pH 4.4), and 0.1 M N-acetylgalactosamine (Sigma Aldrich), the latter to inhibit α-galactosidase B activity (Mayes et al., 1981 (link)). Following a 30 min incubation at 37°C, reactions were terminated by the addition of 480 μL of 0.1 M ethylenediamine, pH 10.3. The amount of 4-methylumbelliferone (4-MU) produced was determined by measuring fluorescence using a Synergy H1 fluorometer (BioTek). Tissue α-Gal A activities were expressed as nmol of 4-MU produced per h per mg of total protein (nmol/h/mg). Measurement of plasma GLA activities in wildtype mice for PK studies was performed as described above with the following modifications: lysates were incubated with 5 mM 4-methylumbelliferyl α-D-galactopyranoside in assay buffer [20 mM citrate, 30 mM sodium phosphate (pH 4.4), 0.1 M N-acetylgalactosamine, and 4 mg/mL BSA], and the reaction was stopped by addition of stop buffer (0.1 M Glycine, 0.1 N NaOH], as previously described (Shen et al., 2016 (link)).
AGA activity was measured with 1 mM L-aspartic acid β-(7- amido-4-methylcoumarin) in 10% SuperBlock and 90% 50 mM Tris-HC (pH 7.5) for 60 min at 37°C, and then adding 100 µL of stop buffer [0.2 M glycine, 0.175 M NaOH (pH 10.6)], as previously described (Mononen et al., 1993 (link)). GUSB enzyme assay was performed using 10 mM 4-methylumbelliferyl-β-D-glucuronide (Merck) in 0.1 M sodium acetate (pH 4.6) at 37°C for 30 min, and reactions were stopped by 0.1 M sodium carbonate (Grubb et al., 2008 (link)). GAA activity assay was performed with 3 mM 4-methylumbelliferyl-a-D-glucopyranoside (Merck) in assay buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, pH 4.0) at 37°C for 3 h (Flanagan et al., 2009 (link)). Reactions were stopped by the addition of an equal volume of 0.4 M glycine, pH 10.8. IDS activity assay was performed with 2.5 mM 4-Methylumbelliferyl sulfate potassium salt (Merck) in 50 mM sodium acetate, at 37°C for 4 h (Dean et al., 2006 (link)). Reactions were stopped with glycine carbonate buffer (pH 10.7). Fluorescence was measured by microplate reader with 360/40 nm excitation and 440/30 nm emission filters.

We performed immunostaining similarly to our previous report (Liu et al, 2021 (link)). In brief, we firstly incubated the zebrafish samples in blocking solution (0.3% Triton X-100, 4% BSA in PBS) at room temperature for at least 1 h. We then incubated the samples in the blocking solution with primary antibodies at 4°C overnight. After removing the primary antibodies, we washed the samples with washing buffer (0.3% Triton X-100 in PBS) 10 times at room temperature for at least 4 h in total. Next, we incubated the samples in the blocking solution with fluorescent dye-conjugated secondary antibodies and the nuclear counterstain DAPI (Thermo Fisher Scientific) at 4°C overnight. Afterwards, we removed the secondary antibodies and DAPI and washed the samples with washing buffer 10 times at room temperature for at least 4 h. The following primary antibodies were used: chicken anti-GFP (1:500, GFP-1020; Aves Labs), goat anti-tdTomato (1:500, MBS448092; MyBioSource), mouse anti-mNeonGreen (1:50, 32F6; ChromoTek), rabbit anti-Insulin (1:100, customised; Cambridge Research Biochemicals), mouse anti-Glucagon (1:50, G2654; Sigma-Aldrich), rabbit anti-Cdh17 (1:1,000; customised sera, gift from Prof. Ying Cao, Tongji University), and rabbit anti-Vasnb (1:1,000; customised sera, gift from Dr. Paolo Panza).
Before confocal imaging, we mounted the stained samples in VECTASHIELD Antifade Mounting Medium (Vector Laboratories) on microscope slides with the pancreas or liver facing the cover slips. We imaged the pancreas and liver with the Leica TCS SP8 platform.

The solid phase AP C3 convertase was built up as described by Hourcade et al. in 2002 (37 (link)) with modifications. C3b was immobilized at 5 µg/ml on microplate wells (Maxisorp, NUNC). After washing, 2 µg/ml FB, 4 µg/ml properdin and 0.1 µg/ml FD were added in convertase buffer (DPBS containing 4% BSA, 2 mM NiCl₂, 0.1% Tween-20) for 1 hour at 37°C. Convertase formation was detected with goat anti-FB and HRP-conjugated rabbit anti-goat antibodies. Convertase activity was determined by adding 10 µg/ml C3 for 1 hour at 37°C. The generated C3a was measured by the C3a EIA kit (Quidel). In some assays, patient’s or control IgG was added together with convertase components to analyze their effect on convertase formation. Convertase decay was determined after incubation of the formed convertase with the isolated IgG fractions alone or in the presence of 1 µg/ml FH for 1 hour at 37°C. Remaining convertase was detected with goat anti-FB antiserum.