PMID- 11027963
OWN - NLM
STAT- MEDLINE
DCOM- 20001207
LR  - 20190702
IS  - 0027-5107 (Print)
IS  - 0027-5107 (Linking)
VI  - 470
IP  - 2
DP  - 2000 Oct 31
TI  - A staining procedure for micronucleus test using new methylene blue and acridine 
      orange: specimens that are supravitally stained with possible long-term storage.
PG  - 103-8
AB  - The micronucleus test has been widely used as an in vivo cytogenetic test. It 
      employs two different kinds of supravital staining methods which use either new 
      methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a 
      new staining procedure for the preparation of specimens supravitally stained with 
      possible long-term storage, using both N and AO. This N/AO-staining method 
      involves three steps; (1) combination of the target tissue or target cells with 
      an equivalent volume of 0.5% solution of new methylene blue (N-staining step), 
      (2) immediate smear of the mixture, followed by treatment with methanol for 10 
      min for fixation and removal of N and drying (referred to as fixed-decolorized 
      specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by 
      washing with Sorensen's buffer (pH 6.8) and covering of specimens before 
      observation (AO-staining step). To examine whether the N/AO-staining method is 
      useful for the micronucleus test, comparisons were made between N-, N/AO-, and 
      AO-stained specimens prepared supravitally from peripheral blood of rats with and 
      without treatment of cyclophosphamide. The results indicate that N/AO-stained 
      specimens can be supravitally observed after long-term storage with the same 
      coloration and comparable frequencies of micronucleated reticulocytes with a 
      positive response as AO-stained specimens, if the staining process is temporarily 
      stopped before AO-staining (as fixed-decolorized specimens), or if the 
      AO-staining step is repeated. The results also showed that separated reticulocyte 
      types are supravitally stained in a similar fashion to N-stained specimens but 
      not to AO-stained specimens, indicative of the preservation of the supravital 
      feature of N-staining. Taken together these results suggest that the 
      N/AO-staining procedure could offer an additional useful staining tool for the 
      micronucleus test.
FAU - Sugihara, T
AU  - Sugihara T
AD  - Department of Drug Safety Research, Kawashima Co., Ltd., 2-1 Takehaya-machi, 
      Kawashima-cho, Hashima-gun, 501-6024, Gifu, Japan.
FAU - Sawada, S
AU  - Sawada S
FAU - Hakura, A
AU  - Hakura A
FAU - Hori, Y
AU  - Hori Y
FAU - Uchida, K
AU  - Uchida K
FAU - Sagami, F
AU  - Sagami F
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - Mutat Res
JT  - Mutation research
JID - 0400763
RN  - 0 (Coloring Agents)
RN  - 0 (Mutagens)
RN  - 8N3DW7272P (Cyclophosphamide)
RN  - F30N4O6XVV (Acridine Orange)
RN  - T42P99266K (Methylene Blue)
SB  - IM
MH  - *Acridine Orange
MH  - Animals
MH  - *Coloring Agents
MH  - Cyclophosphamide/pharmacology
MH  - Male
MH  - *Methylene Blue
MH  - Micronucleus Tests/*methods
MH  - Microscopy, Fluorescence
MH  - Mutagens/pharmacology
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Specimen Handling
EDAT- 2000/10/12 11:00
MHDA- 2001/02/28 10:01
CRDT- 2000/10/12 11:00
PHST- 2000/10/12 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/10/12 11:00 [entrez]
AID - S1383574200000442 [pii]
AID - 10.1016/s1383-5742(00)00044-2 [doi]
PST - ppublish
SO  - Mutat Res. 2000 Oct 31;470(2):103-8. doi: 10.1016/s1383-5742(00)00044-2.