Experimental methods summary (4): tissue-level methods
1. Immunohistochemistry
1.1 SP method
1.2 Double-antibody sandwich ELISA
1.3 Colloidal gold method
2. Hematoxylin and eosin staining
3. In situ hybridization
MMP-2 and MMP-9 monoclonal antibodies and an SP kit were obtained from ZYMED (the original text gives “1 100”). Use 3% H2O2 to block endogenous peroxidase, retrieve antigens by microwave heating, incubate in 10% normal goat serum, add primary antibody and leave overnight at 4°C. Wash with PBS (Na2HPO4 8.1 mmol/L,KH2PO4 1.5 mmol/L, NaCl 137 mmol/L, 2.7 mmol/L, pH 7.4) with agitation; add secondary and tertiary antibodies in sequence, develop with DAB, counterstain with hematoxylin and mount routinely.
A double-antibody sandwich ELISA used purified polyclonal antibody for capture and laboratory-prepared anti-LM Internalin A monoclonal antibody 3R6 for detection. The sequence was: diluted polyclonal antibody → three washes → 3% BSA block → three washes → diluted test bacteria → three washes → diluted monoclonal antibody → four washes → enzyme-labeled goat anti-mouse IgG → five washes → TMB substrate → stop solution → OD450 measurement. Antibody, antigen and BSA diluents were 0.01 mol/L PBS at pH 7.4; the wash solution was 0.01 mol/L PBS at pH 7.4 with 0.1% Tween-20 (PBST). The original text defines a positive result as (test-well OD − blank-control OD) / (negative-control OD − blank-control OD) ≥ 2.1 (P/N ≥ 2.1).
① Glassware preparation: soak flasks and other glassware for 48 hours in acid prepared from potassium dichromate and concentrated sulfuric acid; rinse repeatedly with distilled water, rinse three or four times with ultrapure water, and dry. ② Colloidal gold preparation: the cited method uses trisodium citrate reduction. Add 1 mL of 1% chloroauric acid to 99 mL ultrapure water in a clean flask to make 100 mL of 0.01% solution. Heat to boiling with continuous magnetic stirring; quickly add 1% trisodium citrate, keep stirring until the color becomes purplish red, reflux for another 10 minutes, cool to room temperature and store at 4°C. ③ Labeling an anti-clenbuterol monoclonal antibody: place an appropriate amount of prepared colloidal gold in a beaker and use 0.2 mol/L K2CO3 solution to adjust the colloidal gold to pH 6.0. Dissolve antibody in ultrapure water at one tenth of the colloidal gold volume, add dropwise and stir for 30 minutes (labeling concentrations 0.5, 1 and 2 μg/mL). Add 10% BSA dropwise at one tenth of the colloidal gold volume and block with stirring for 30 minutes; then add 1% PEG 20000 at one tenth of the volume and block with stirring for another 30 minutes. Centrifuge at 9,000 rpm for 30 minutes, remove the supernatant and add reconstitution solution at one tenth of the original volume (1% BSA, 2% sucrose, 0.05% PEG 20000 and 0.1% NaN3 in 0.02 mol/L phosphate buffer, pH 7.4) to resuspend the gold-labeled antibody. ④ CLE strip immunoreaction kinetics: add CLE standards to pooled negative pig urine at 0, 0.2, 0.5 and 1.0 ng/mL; apply 70 μL to each strip. Read the T and C line values and T/C ratio every 30 seconds for 30 minutes, plot each against time and observe changes with concentration and time. The original article uses these kinetic curves as indirect measures of antigen–antibody reactions on the strip.
1. Deparaffinization: xylene I for about 10 minutes (10 minutes on an automated stainer); xylene II and III for about 5 minutes (10 minutes automated); absolute ethanol I and II for 1 minute each; 95% ethanol for 1 minute; 85% ethanol for 1 minute; then a brief tap-water rinse (1 minute automated). 2. Staining: hematoxylin for about 1–5 minutes; brief tap-water rinse; differentiate in 1%, 0.5% or 0.25% aqueous hydrochloric acid for 3–5 seconds (30 seconds automated); rinse in tap water and briefly blue in warm water (5 minutes automated); counterstain with alcoholic eosin for 20 seconds to 2 minutes (30 seconds to 5 minutes automated). 3. Dehydrate, clear and mount: 85% ethanol for 20 seconds; 95% ethanol for 1 minute; absolute ethanol I for 1–2 minutes and II for 2 minutes; xylene I and II for 2 minutes each; mount with neutral resin or Canada balsam.
Dual staining for miR-129-5p and type I collagen or integrin α1 was performed on the same section using locked-nucleic-acid fluorescence in situ hybridization (FISH) and immunofluorescence. Dry frozen sections thoroughly, rinse in DEPC-PBS, soak in 0.3% Triton X-100 for 10 minutes, digest with 20 μg/mL proteinase K for 5 minutes and rinse twice in DEPC-PBS for 5 minutes each. Prehybridize for 1 hour at 60°C in hybridization solution (SSC, blocking reagent, formamide, NLS and SDS), add 20 nM miR-129-5p LNA probe, denature at 83°C for 5 minutes and incubate at 53°C for 20 hours. Wash twice for 20 minutes in wash buffer (SSC, formamide and NLS) preheated to 60°C; incubate in RNase buffer at room temperature for 5 minutes, then in RNase buffer containing 20 μg/mL RNase A at 37°C for 30 minutes. Wash twice in 2× SSC/0.1% NLS at 37°C for 20 minutes each, then twice in 0.2× SSC/0.1% NLS under the same conditions. Incubate in TBS (blocking reagent, Tris-HCl and NaCl) for 1 hour at room temperature, add POD-DIG and the immunofluorescence primary antibody (type I collagen or integrin α1), and incubate overnight at room temperature. Rinse twice for 10 minutes at room temperature in TS9.5 (Tris-HCl, NaCl and MgCl2), treat with TSA™ Biotin System for 30 minutes, add Alexa 488-labeled goat anti-rabbit IgG and Cy3-avidin and incubate for 4 hours at room temperature in the dark. Counterstain with DAPI, rinse three times in TNT solution for 5 minutes each, mount in glycerol and observe by confocal laser microscopy.
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