Five micron-sections of lung tissue or reconstituted ALI epithelium, fixed in 4% formaldehyde and paraffin-embedded, were deparaffinised in toluene and rehydrated through a graded series from ethanol to water. Antigen retrieval was performed in citrate buffer (pH 6• 0 containing 0• 1% of triton) using a pressure cooker at 15 PSI for 5 min. Sections were blocked for non-specific antigen binding by incubation in Bloxall (Vector Laboratories Inc.) for 15 min and then in 0• 3% hydrogen peroxide with 5% goat serum (Bio-Rad) for 30 min. Staining first included a 30 min protein blocking with 5% goat serum, then the primary antibodies diluted in 5% normal goat serum solution were applied, then the appropriate SuperBoost™ goat anti-rabbit or anti-mouse, poly-HRP-conjugated secondary antibody (Thermo Fisher Scientific) was applied for 40 min. HRP-conjugated polymer mediated the focal covalent binding of a fluorophore using tyramide signal amplification. Table S5 recapitulates the antibodies and fluorophores that were used. Finally, sections were counterstained with Hoechst (Thermo Fisher Scientific) diluted at 10 µg/ml in TBS-BSA 5% and mounted with Dako fluorescence mounting medium (Dako, Carpinteria, CA). For negative controls, we used rabbit or mouse isotype controls at the same concentration as the corresponding primary antibodies (diluted in 5% normal goat serum).
Goat serum
Manufactured by Bio-Rad
Sourced in Germany
Goat serum is a laboratory product derived from the blood of healthy goats. It is used as a supplement in cell culture media to support the growth and maintenance of various cell lines. Goat serum provides a complex mixture of proteins, growth factors, and other essential nutrients required for cell proliferation and function.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum, by Thermo Fisher Scientific (2 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (2 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
β amyloid, by BioLegend (1 mentions)
Trueblack, by Biotium (1 mentions)
Fv1000 spectral confocal microscope, by Olympus (1 mentions)
Donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Goat serum, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Gfp a 11122, by Thermo Fisher Scientific (1 mentions)
Pbs t, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
6 protocols using goat serum
1
Immunohistochemistry on Lung Tissue
2
Immunostaining of Neuronal Markers
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Tissue sections stained by mRNA FISH and fixed primary neurons were washed with 1× PBS and blocked in 1× PBS supplemented with 5% (v/v) goat serum (Bio-Rad), 1% (wt/vol) BSA (Sigma) and 0.1% (v/v) Triton X-100 (Sigma) at RT for 1 h. Primary antibodies were diluted in blocking solution and applied to samples at 4°C overnight. Primary antibodies used in this study were: βAmyloid (BioLegend, 803001, RRID:AB_2564653 , 1:200), GFAP (Sigma, G3893, RRID:AB_477010 , 1:500), Homer (SynapticSystems, 160003, RRID:AB_887730 , 1:500), Iba1 (Fujifilm Wako, 019-19741, RRID:AB_839504 , 1:250), misfolded SOD1 (Médimabs, MM-0070-P, RRID:AB_10015296 , 1:100), vGlut (MerckMillipore, AB5905, RRID:AB_2301751 , 1:300); negative controls omitted the primary antibody. This was followed by 4× 10 min washes in 1× PBS at RT and subsequent application of suitable goat Alexa Fluor Plus secondary antibodies (488/546/647) diluted 1:500 in blocking solution at RT for 2 h. Samples were then washed 4× 10 min with 1× PBS at RT. Cryosections only were treated with 1× TrueBlack (Biotium) at RT for 30 s to quench autofluorescence caused by the accumulation of lipofuscin and other protein aggregates, followed by 2× washes with 1× PBS. Nuclei of samples were counterstained with DAPI (Sigma; shown in blue in all confocal images) at 1 μg/ml in PBS and samples were mounted using DAKO Fluorescence Mounting Medium (Agilent).
3
Immunocytochemistry of TM9SF4 Protein
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5x104 cells were fixed in formaldehyde 3.7% and permeabilized with 0.5% PBS-triton X-100 then plated by cytospin (Cytospin3, SHANDON) on polylysine-coated microscope slides. After blocking with PBS-triton 0.1%-goat serum (Biorad) 3% (15 minutes, room temperature), cells were stained over-night at +4°C with TM9SF4 primary antibody (1:50) (S-20, Santa Cruz). Subsequently, slides were washed 3–4 times with PBS, incubated with AlexaFluor-647 (1:1000) (donkey anti–goat IgG, Invitrogen) for 1 hour at room temperature, washed again and stained with 4’,6-Diamidino-2-Phenylindole (DAPI) to label the nuclei (10 minutes, room temperature). Fluorescence was detected using an Olympus FV-1000 spectral confocal microscope.
4
IFA Protocol for Detecting FCoV Antibodies
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The IFA procedure in this study was based on previously published protocols, with modifications [13 (link),15 (link)]. The effusion samples were processed and stored at 4 °C on the day they arrived. First, the samples were washed with PBS, and the cell count was adjusted to 200,000–500,000 cells per 100 µL. The processed samples were then centrifuged at 1000 rpm for 10 min at 4 °C with a cytocentrifuge. The slides were then fixed with 80% acetone at −20 °C. For blocking, the slides were incubated with 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature in a moist box. The mouse anti-FCoV N protein monoclonal antibody (Bio-Rad, MCA2194) was used at a 1:400 dilution in 10% goat serum for 45 min at room temperature as the primary antibody. The slides were washed three times with PBST (PBS containing 0.01% Tween 20), and goat anti-mouse IgG FITC conjugate (Jackson ImmunoResearch) was then added at a 1:400 dilution in 10% goat serum for 30 min at room temperature in the dark as the secondary antibody. Finally, the slides were washed for 15 min and sealed with mounting solution containing DAPI (Vectashield, Burlingame, CA, USA). The slides were then interpreted with a fluorescence microscope (Olympus IX83).
5
Immunohistochemical Staining of Fly Antennae
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Flies were anesthetized and the antenna dissected. The antennae were fixed in 2% paraformaldehyde (PFA, Electron microscopy studies) in phosphate-buffered saline with 0,5% Triton X-100 (PBST, Sigma-Aldrich) for 55 min at RT and subsequently washed with PBST for 4 × 10 min at RT on a shaker.
Blocking was done using 5% goat serum (Thermofisher) in PBST for 1.5 h at RT and primary antibody was added (GFP A11122 or A11070 Invitrogen, HA-Tag C29F4 Cell signaling technology) diluted 1:300 in 5% goat serum in PBST. The primary antibody is incubated for 4 h on a shaker at RT for 4 h and afterwards for 48 h on 4 °C.
After incubation the samples are washed in PBST 5 × 15 min on a RT shaker secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG A11008 Invitrogen, V5-TAG:DyLight550 MCA1360D550GA Biorad,) diluted 1:500 in 5% goat serum in PBST is added to the samples. The secondary antibody is incubated for 4 h at RT and afterwards for 48-72 h on 4 °C. Samples are washed in PBST 5 × 15 min on a shaker at RT and afterwards mounted with RapiClear (SunJin Lab, Hsinchu, Taiwan, RC149002). Imaging was performed on a Leica SP8 confocal microscope with a 63x, 1.4 NA objective at a resolution of 512 × 512.
Blocking was done using 5% goat serum (Thermofisher) in PBST for 1.5 h at RT and primary antibody was added (GFP A11122 or A11070 Invitrogen, HA-Tag C29F4 Cell signaling technology) diluted 1:300 in 5% goat serum in PBST. The primary antibody is incubated for 4 h on a shaker at RT for 4 h and afterwards for 48 h on 4 °C.
After incubation the samples are washed in PBST 5 × 15 min on a RT shaker secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG A11008 Invitrogen, V5-TAG:DyLight550 MCA1360D550GA Biorad,) diluted 1:500 in 5% goat serum in PBST is added to the samples. The secondary antibody is incubated for 4 h at RT and afterwards for 48-72 h on 4 °C. Samples are washed in PBST 5 × 15 min on a shaker at RT and afterwards mounted with RapiClear (SunJin Lab, Hsinchu, Taiwan, RC149002). Imaging was performed on a Leica SP8 confocal microscope with a 63x, 1.4 NA objective at a resolution of 512 × 512.
6
BDNF Protein Extraction and Detection
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Hippocampi were lysed in extraction buffer (50 mM sodium acetate, 1M NaCl, 0,1% Triton X100, pH 4,0, protease inhibitor tablets), and by means of a Sonifier (UP50H Hielscher) with a MS1 sonotrode. Cell lysates were cleared at 20.000× g for 10 min at 4°C. Proteins were blotted on Immun-Blot PVDF membranes (Biorad). Blocking and antibody incubation were performed in 10% goat serum, 5% milk powder (Biorad) for 3–4 h. For BDNF detection, rabbit anti-BDNF 17H (1/6000, provided by Michael Sendtner, Institute for Clinical Neurobiology, Würzburg, Germany) was incubated over night at 4°C. Anti-Cytochrome C A-8 (Santa Cruz, 0.2 μg/ml) was used as loading control. For detection, the ECL Plus kit (GE Healthcare) was used in combination with horseradish peroxidase-coupled secondary antibodies (Jackson Laboratories).
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