2.5 Blood and Tissue
Collection
1. 100–200 μl capillary tubes.
2. Ophthalmic ointment and cotton balls for post-bleeding
hemostasis.
3. Precision balance.
4. Isoflurane and isoflurane vaporizer.
5. Sterile scalpels, scissors, and forceps.
6. 5 ml syringes with 25 G Â 5/8
00 needle.
7. 5 ml EDTA tubes (BD Vacutainer).
2.6 ELISA
1. 96-well microtiter plates.
2. Purified protein (diluted to 1 μg/100 μl PBS).
3. Multichannel pipettors or 96 well automated washer.
4. Protein-free blocking buffer (PFB) (Pierce).
5. Horse radish peroxidase (HRP)-conjugated goat anti-Syrian
hamster immunoglobulin G (IgG) diluted in PFB (1:5000).
6. 3,3
0 ,5,5
0 -tetramethylbenzidine (TMB) substrate solution.
7. ELISA plate reader.
8. Pipettors and sterile barrier pipette tips.
2.7 Tissue
Colonization
1. 15 ml tube with 10 ml semisolid EMJH supplemented with
100 μg/ml 5-fluorouracil (5-FU): Add 1.5 g of Noble agar to
880 ml EMJH basal medium and autoclave. Cool to ~50
C
and then add 100 ml of EMJH supplement, 1 g of lactalbumin
hydrolysate, 1 mg of SOD, 40 mg of sodium pyruvate, 10 ml of
heat-inactivated rabbit serum, and 10 ml of 0.01 g/ml 5-FU,
sterilized through a 0.22 μm filter. Prepare 10 ml aliquots in
15 ml tubes and store at 4
C.
2. Dark-field microscope.
3. Tissue homogenizer/disruptor.
4. DNeasy Blood and Tissue Kit (Qiagen).
5. Spectrophotometer.
6. qPCR master mix (Bio-Rad iTaq Universal Probes Supermix).
7. Primers at the concentration of 10 μM and probe at the concentration of 5 μM [15].
Name
Oligonucleotides (5
0
! 3
0 )
LipL32-45F
AAG CAT TAC CGC TTG TGG TG
LipL32-268F
GAA CTC CCA TTT CAG CGA TT
LipL32-189P
FAM AA AGC CAG GAC AAG CGC CG BHQ1
8. Quantitative thermal cycler.
Leptospirosis Vaccine Studies in Hamsters
261
Collection
1. 100–200 μl capillary tubes.
2. Ophthalmic ointment and cotton balls for post-bleeding
hemostasis.
3. Precision balance.
4. Isoflurane and isoflurane vaporizer.
5. Sterile scalpels, scissors, and forceps.
6. 5 ml syringes with 25 G Â 5/8
00 needle.
7. 5 ml EDTA tubes (BD Vacutainer).
2.6 ELISA
1. 96-well microtiter plates.
2. Purified protein (diluted to 1 μg/100 μl PBS).
3. Multichannel pipettors or 96 well automated washer.
4. Protein-free blocking buffer (PFB) (Pierce).
5. Horse radish peroxidase (HRP)-conjugated goat anti-Syrian
hamster immunoglobulin G (IgG) diluted in PFB (1:5000).
6. 3,3
0 ,5,5
0 -tetramethylbenzidine (TMB) substrate solution.
7. ELISA plate reader.
8. Pipettors and sterile barrier pipette tips.
2.7 Tissue
Colonization
1. 15 ml tube with 10 ml semisolid EMJH supplemented with
100 μg/ml 5-fluorouracil (5-FU): Add 1.5 g of Noble agar to
880 ml EMJH basal medium and autoclave. Cool to ~50
C
and then add 100 ml of EMJH supplement, 1 g of lactalbumin
hydrolysate, 1 mg of SOD, 40 mg of sodium pyruvate, 10 ml of
heat-inactivated rabbit serum, and 10 ml of 0.01 g/ml 5-FU,
sterilized through a 0.22 μm filter. Prepare 10 ml aliquots in
15 ml tubes and store at 4
C.
2. Dark-field microscope.
3. Tissue homogenizer/disruptor.
4. DNeasy Blood and Tissue Kit (Qiagen).
5. Spectrophotometer.
6. qPCR master mix (Bio-Rad iTaq Universal Probes Supermix).
7. Primers at the concentration of 10 μM and probe at the concentration of 5 μM [15].
Name
Oligonucleotides (5
0
! 3
0 )
LipL32-45F
AAG CAT TAC CGC TTG TGG TG
LipL32-268F
GAA CTC CCA TTT CAG CGA TT
LipL32-189P
FAM AA AGC CAG GAC AAG CGC CG BHQ1
8. Quantitative thermal cycler.
Leptospirosis Vaccine Studies in Hamsters
261