In Situ Detection of Bacteria in Mouse Gingival Tissue Sections

Samenvatting

Source: Choi, Y. S., et al. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA. J. Vis. Exp. (2015).

This video demonstrates the in situ detection of bacteria in mouse gingival tissue sections using digoxin-labeled DNA probes targeting bacterial DNA. The section is incubated with the probe. Alkaline phosphatase-conjugated antibodies specific to digoxin are added. Endogenous phosphatases are inactivated, and a colored precipitate is obtained by substrate addition. The section is counterstained, dehydrated, cleared in xylene, and mounted before microscopic visualization of bacteria.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Probe Preparation

1. PCR amplification of the probe
   1. For the Porphyromonas gingivalis–specific probe, amplify a 343-bp DNA fragment of P. gingivalis 16S rRNA by PCR using the genomic DNA of P. gingivalis and the following primers: 5'- TGC AAC TTG CCT TAC AGA GG-3' and 5'- ACT CGT ATC GCC CGT TAT TC-3'. Perform amplifications with the following cycle conditions: 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min 20 s, followed by a 5 min ext....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Acetic anhydride	Sigma	6404
50% Dextran sulfate solution	Millipore	S4030
50X Denhardt’s solution	Sigma	D2532
Diethyl pyrocarbonate (DEPC)	Sigma	P159220
Digoxigenin (DIG) DNA labeling and detection kit	Roche	11 093 657 910
Formamide	Sigma	F9037
ImmEdge™ Pen	Dako	H-400
Levamisole	Vector	SP-5000
Magnesium chloride	Sigma	246964
Maleic acid	Sigma	M0375
Methyl green	Sigma	M6776
Paraformaldehyde	Sigma	P1648
Permount	Fisher	SP15-500
Salmon sperm DNA solution	Invitrogen	#15632-011
Sodium chloride	Sigma	S9625
Sodium citrate	Duksan	D1420
Sodium dodecyl sulfate	Amresco	227
Triethanolamine-HCl	Sigma	90279
Tris-HCl	Research organics	3098T