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General Information
 | The ImmnoHistoWax process (IHW process) is a new fixation and embedding
method for immunohistochemistry, which preserves both morphology nad antigen
immunoreactivity. IHW is of intrest for immunology and expérimental pathology,
when preservation of antigen and better morphology than what is attainable by
cryostat sections is needed |
| All processing and sectioning steps are closely related to
formalin-fixation and parrafin embeding procedure. ImmunoHistoWax process
requires few changes in laboratory practices, and may be performed with
standard equipment for routine Histology. |
| ImmunoHistoWax process is a method based on minimal denaturation of
protein during fixation and embedding. This is achieved by a strong
stabilisation of proteine structure avoiding further chemical denaturation
induced by organic (dehydrating) solvents and by embedding in an inert wax at
37°C. The process has been successfully tested under several expérimental
conditions, including imunostaining of membrane or intracellular proteins
withmonoclonal or polyclonal antibodies, staining of carbohydrate residues
with lectins, detection of receptor with their natural ligands and
identification of apoptotic cells.
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Reference |
Protocol
| Tissue samples are fixed in ImmunoHistoFix ( an aldehyde - free zinc
fixative) for 3 days at 4°C. Sample thickness should not exceed 5 mm to allow
optimal infiltration of fixative and dehydrating agents. |
| Dehydration is performed by two different protocols : the samples where
either dehydrated in a graded series of ethanol bathes : 30%, 50%, 70%, 90%
and 100% for 30 minutes each at room temperature, or samples where dehydrated
in acetone for 6 hours. The first protocol appears to preserve better tissular
morphology. The second protocol may be less cumbersome, and requires less
attention to timing. |
| Infiltration is performed at 37°C by 3 bathes of ImunoHistoWax ( 20
minutes each ) |
| Tissue specimens are then embedded in ImmunoHistoWax. Embedded samples me
be stored for at least one year at room temperature without loss of
antigenreactivity or morphological structure. |
| Samples are sectioned with rotary or sliding microtomes. The cutting
procedure of ImmunoHistoWax embedded specimens permets reproducible serial
sectioning. 3-5 µm sections were transferreddirectly using thn grids on a drop
of water on precoated (gelatin, poly-L-lysin,..) slide. Floating onwather bath
is onsuitable because of the slight hydrophilicity of the wax. Slides are air
drid at room temperature and stored at room temperature for up to several
months. |
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Tissue sample (one dimension should not exceed 5 mm)
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Fixation (immersion in fixative soluton for 3 days at 4°C)
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Dehydration:
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Ethanol (for the best morphology graded serie of 30%, 50%, 70%, 90%, 100% for 30 minutes each bath.
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Acetone (one bath of aceton for 6 hours)
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Embedding (ImmunoHistoWax
infiltration by 3 bathes for 20 minutes each at 37°c
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Sectionning 3 to 5µm
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Immunostaining
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Single Staining and double
Staining.
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