Goat anti rabbit immunoglobulins hrp

Mouse liver specimens were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4; 1% Triton X-100; 25 mM HEPES; 150 mM NaCl; 0,2% SDS; 5 mM MgCl₂; 1 mM Na₃VO₄; 1 mM NaF) containing protease inhibitors (Roche, #04 693 132 001) using an Ultra-Turrax® homogenizer. After 40 min in ice, samples were centrifuged at 15,000 g. Proteins from diluted supernatant were assayed with the Bradford method (BioRad). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with non-fat milk in TBS (20 mM Tris, 137 mM NaCl) during 1–2 h and incubated overnight at 4 °C with anti-cleaved caspase-3, anti-RIPK1 (Cell Signaling, 3493) anti-actin (Sigma A3854), anti-phospho-Thr183/Tyr185-JNK (Cell Signaling, 9251) or anti-JNK (Calbiochem, 559304) primary antibodies, and then with secondary goat anti-rabbit immunoglobulins/HRP (Dako, P0448). Protein-antibody complexes were revealed by enhanced chemiluminescence (Millipore) and ImageQuant LAS-4000 mini imager analysis (GE-Healthcare). The Multi Gauge software was used for signal quantifications and data was expressed as levels relative to signals detected in PBS controls. Cleaved caspase-3 expression levels or JNK phosphorylation status were respectively normalized on β-actin expression levels or on total JNK expression levels.

The tumor tissue was fixed with 4% paraformaldehyde, sliced in 4 μm sections and blocked with protein block serum-free (X0909, Dako, Jena, Germany). The expression of the metabolic targets was analyzed by immunohistochemistry using anti-rabbit PDH E1 alpha polyclonal antibody (18068-1-AP, dilution 1:500, Proteintech, Rosemont, IL, USA), anti-mouse monoclonal antibody OGDH (66285-1-Ig, dilution 1:100, Proteintech), anti-rabbit LDHA polyclonal antibody (19987-1-AP, dilution 1:100, Proteintech), anti-rabbit anti-MCT-4 antibody (bs-2698R, dilution 1:100, Biossusa, Woburn, MA, USA). Goat anti-rabbit Immunoglobulins/HRP (P0448, Dako) or goat anti-mouse Ig/HRP antibody (P40447, Dako) were used as secondary antibodies.

Rhodamine-phallotoxin was purchased from Molecular Probes (Eugene, OR, USA). Mouse anti-human FHL2 used for western blot were a product of the MBL international incorporation (11–134, MBL International Incorporation, Woburn, Japan). Rabbit anti-human FHL2 antibody was used for IHC and was purchased from Abcam (Cambridge, UK). Mouse anti-FOXK1 (G-4), Vimentin (E-5), E-Cadherin (H-108), GAPDH (G-9) and bovine anti-mouse IgG-TR and goat anti-rabbit IgG-FITC were purchased from Santa Cruz (Santa Cruz, CA, USA). Mouse anti-human Flag were a product of Sigma (St Louis, MO, USA). Goat anti-rabbit immunoglobulins/HRP, rabbit anti-mouse immunoglobulins/HRP, normal mouse and rabbit IgG were all products of Dako (Carpinteria, CA, USA).
Human colon cancer cell lines SW480, Caco2 and SW620 were all maintained in our laboratory as previously described.^{26 (link), 27 (link)} The cells were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin in a humidified incubator at 37 °C with an atmosphere of 5% CO₂.

The formalin‐fixed paraffin‐embedded lung tissue sections (4.25 μm in thickness) were stained with hematoxylin and eosin (H&E). An immunohistochemistry assay was employed using several primary antibodies against SARS‐CoV‐2 spike protein (mouse, GTX632604, GeneTex), SARS‐CoV‐2 nucleocapsid protein (rabbit, GTX635679, GeneTex), CD31 (rabbit, ab28364, Abcam), CD41 (rabbit, ab134131, Abcam), CD45 (rat, #103202, Biolegend), MPO (rabbit, ab9535, Abcam), CD68 (rabbit, ab125212, Abcam), and P‐selectin/CD62P (rabbit, ab255822, Abcam). The used secondary antibodies include Goat Anti‐Rabbit Immunoglobulins/HRP (P0448, Dako), Goat Anti‐Mouse Immunoglobulins/HRP (P0447, Dako), and Rabbit Anti‐Rat Immunoglobulins/HRP (P0450, Dako). The liquid DAB⁺ Substrate Chromogen System (K3468, Dako) was used for color development. DAB signal‐positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH). The area with a particularly high (N) protein positive signal was defined as “N protein signal‐rich area,” and the positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH).

Proteins were separated by 10 % SDS-PAGE and transferred to PVDF membranes. For blocking, membranes were soaked in 5 % non-fat dried milk in TBS-Tween20 (0.01 %). Proteins were immunodetected using primary antibodies. Then the membranes were washed and incubated with a secondary HRP-coupled antibody. Finally, HRP signal was revealed using the Immobilon Western Chemiluminescent HRP Substrate (ref. WBKLS0100; Merck Millipore). The intensity of the bands was densitometrically quantified using the Image J software (v. 1.45s).
Primary antibodies: mouse anti-CD9 (1:250; ref. 555370, BD Biosciences), mouse anti-CD63 (1:1000; ref. OP171, Calbiochem), mouse anti-CD81 (1:1000; ref. sc-166028, Santa Cruz), mouse anti-TSG101 (1:500; ref. Ab83, Abcam), mouse anti-Flotillin-1 (1:250; ref. 610821, BD Biosciences), rabbit anti-Annexin V (1:1000; ref. ab108321, Abcam) and mouse anti-Haptoglobin (1:1000; ref. ab13429, Abcam). Secondary antibodies: rabbit anti-mouse Immunoglobulins/HRP, 1:2000, ref. P0260, Dako; and goat anti-rabbit Immunoglobulins/HRP, 1:2000, ref. P0448, Dako. Bands’ intensity was quantified using the Image J software (v. 1.45s).

Localization and expression of FAM13A protein was determined using immunohistochemical staining in lung tissue. Lung tissue was embedded in paraffin and cut into three-μm thick lung sections. Antigen retrieval was done with 10 mM citrate buffer at pH 6.0 followed by avidin/biotin blocking using the Avidin/Biotin blocking kit (Vector Laboratories, Inc., Burlingame, CA 94010). Specific primary antibody against FAM13A (55401-1-AP, Proteintech, Manchester, United Kingdom), E-cadherin (610182, BD Bioscience, Breda, NL), secondary antibody Goat Anti-Rabbit Immunoglobulins/HRP (Dako P0488, Amsterdam, NL), Rabbit Anti-Mouse Immunoglobulins/HRP (Dako P0260, Amsterdam, NL) and tertiary antibody Streptavidin/HRP (Dako P0397, Canada) were used. Positive staining was visualized using VECTOR^® NovaRED^TM (SK-4800, Vector Laboratories, Canada). The slides were scanned with the Hamamatsu NanoZoomer 2.0HT digital slide scanner, and representative pictures were taken using the Hamamatsu NDP view 2 software.