Human/Mouse/Rat Total HSP70/HSPA1A DuoSet IC ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.	-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Upstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Publication protocol

At 0 (sham, n = 5), 0.08, 0.5, 2, 6, 12, and 24 h after sonication, rats (n = 5 per time point) were deeply anesthetized with isoflurane before decapitation. The brains were dissected from the skull, and the sonicated tissue and the corresponding sham tissue were isolated. Tissue was snap-frozen in liquid nitrogen and kept at −80 °C until homogenization in cell lysis buffer (9803S; Cell Signaling Technology) containing a protease inhibitor mixture (S8820-2TAB; Sigma-Aldrich). Samples were centrifuged at 15,996 × g for 20 min at 4 °C, and the supernatant was used for analyses. Total protein was determined using a bicinchoninic acid (BCA) assay (23227; Thermo Scientific). Homogenates (2 mg/mL total protein) were analyzed by Rat Bio-Plex Cytokine 24-Plex group I assay (171-K1001M; Bio-Rad Laboratories, Inc.) or ELISAs for the following: stromal cell-derived factor 1 (SDF1) (RS0074; NeoBiolab), BDNF (ELR-BDNF; RayBiotech, Inc.), MMP9; FGF (MFB00), ICAM (RIC100), HSP70 (DYC1663-2), and hepatocyte growth factor (HGF) (MHG00), all from R&D Systems, Inc. All assays were performed according to the manufacturer’s protocols. Samples were run with a protein concentration of 2 mg/mL and were read on a spectrophotometric plate reader (Spectra Max M5; Molecular Devices).

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Manufacturer protocol

Download the product protocol from R&D Systems for Human/Mouse/Rat Total HSP70/HSPA1A DuoSet IC ELISA below.

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