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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.   
• References

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.

 

• Tissue sample (one dimension should not exceed 5 mm)

• Fixation (immersion in fixative soluton for 3 days at 4°C)

• Dehydration:

  • Ethanol (for the best morphology graded serie of 30%, 50%, 70%, 90%, 100% for 30 minutes each bath.

  • Acetone (one bath of aceton for 6 hours)

• Embedding  (ImmunoHistoWax infiltration by 3 bathes for 20 minutes each at 37°c

• Sectionning 3 to 5µm

Immunostaining

• Single Staining and double Staining.

• FloraStain