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To induce active Heymann's glomerulonephritis, maternal kidneys were isolated and homogenized, freed from connective tissue and mixed with complete Freund's adjuvant (1 1) [22 24].
According to their protocol, adult rabbits were injected with a minimum of 100 µg antigen mixed with Complete Freund's Adjuvant subcutaneous and/or intramuscularly at multiple sites.
After centrifugation two times, the eluted protein in the supernatant was quantified and 0.2 mg of protein was mixed with complete Freund's adjuvant and injected subcutaneously into a rabbit.
Female New Zealand White rabbits were first immunized by subcutaneously injecting them with 1 ml of the immunogen (1 mg of rhTNF-α in phosphate-buffered saline (PBS) mixed with complete Freund's adjuvant (Sigma)).
The rats were first immunized by subcutaneously injecting them with 0.5 ml of the immunogen (0.5 mg of KLH-YG1 or KLH in PBS mixed with complete Freund's adjuvant).
The SIINFEKL peptide was thoroughly mixed with complete Freund's adjuvant.
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Briefly, the mice were primary-immunized with 10 µg of H5N1-M2e pre-mixed with complete FA or SAS, and then boosted with 10 µg of the peptide with incomplete FA or SAS twice in a three-week interval.
The eluted proteins were mixed (1∶1) with complete Freund's adjuvant and then used to generate antisera 6F44 and 43C9 against Dr prominin-1a and prominin-1b, respectively, by immunizing New Zealand white rabbits.
All three peptides were further diluted into phosphate-buffered saline each at 2 mg/ml and mixed 1 1 with complete adjuvant containing 8 mg/ml heat-killed M. tuberculosis H37Ra (Difco) in incomplete adjuvant (Imject; Thermo-Fisher) [ 14].
Transformed spheroplasts were mixed with synthetic complete (SC) top agar (1 M sorbitol, 1.92 g/L synthetic complete uracil dropout supplement, 6.7 g/L yeast nitrogen base, 2% glucose, and 2.5% agar) and overlaid onto SC uracil dropout plates.
Five imputed datasets were created and the command mim was used to run mixed logistic and mixed regression models with complete datasets similar to the models described above.
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