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Processing Procedures for JB-4 Resin

By Irene Murray,2014-07-09 19:41
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Processing Procedures for JB-4 Resin

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    Processing Procedures for JB-4 Resin

    A. Fixation

    Fix samples with McDowell’s and Trump’s 4Formaldehyde:1Gluteraldehyde fixative. Do not use osmium (or Bouin’s fixative; the picric acid will soften the plastic).

B. Rinse

     2X (10 min each) in 0.1M Phosphate Buffer (pH 7.2-animal, pH 6.8-plant).

C. Dehydration

     30% ethanol 15 min

    50% ethanol 15 min

    70% ethanol 15 min

    95% ethanol 15 min

    100% ethanol 30 min (X2)

D. Infiltration

    1. Mix up one batch of catalyzed infiltration resin:

    100 mL of JB-4 Solution A

    Add 0.90 g of dry catalyst C; mix until dissolved.

    It helps to place the dry catalyst into a weighing dish and to mix it with a small amount of

    Solution A to form a paste before adding the rest of Solution A, putting the solution in a

    Nalgene bottle and mixing with a stirring bar for 30-60 min. Make sure that the catalyst is

    in solution before proceeding.

    Note: The solution may be stored for several weeks at approximately 4 ? C in the dark.

    2. Infiltration:

     Put samples in infiltration medium at room temperature on rotator. Change to new

    medium after 2-3 hrs. Do third change of medium and leave overnight.

D. Embedding

    1. Mix up one batch of embedding medium:

    1.25 ml of JB-4 Solution B (25% more than recommended.)

    25 ml of fresh catalyzed Solution A.

    Note: Never use exhausted infiltration medium for embedding.

    Stir mixture thoroughly and place into ice bath while arranging samples

    in molds to prevent premature polymerization.

2. Place samples in embedding molds.

    Place samples in BEEM capsules or plastic flat embedding molds (not silicon rubber) and

    fill molds with embedding medium. Capsules must be filled entirely with embedding

    medium Air will prevent proper polymerization so capsules must be filed completely and

    capped or capsules and molds should be put into a RT oven or desiccator with nitrogen

    back-fill. It is advisable to put capsules or molds on a cold surface to minimize the adverse

    effects of the exothermic reaction that takes place when the resin polymerizes. This is

    especially important if the blocks are going to be used for immunocytochemical

    localization.

E. Sectioning

    1. Trim off excess plastic with a razor blade but be sure to leave some

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    excess around all the edges because they will curl slightly.

    2. Break triangular glass knife, orient at ~10? and cut 2-3 μm thick sections.

    Note: sections may need to be much thicker to see some features. For instance, 8µm works well if you want to see gus staining).

    3. Pick up dry sections with forceps and release onto surface of slightly warm water in staining dish (adding a drop of ammonium hydroxide may help the sections spread). 4. Pick up several sections on glass slides and heat on hot plate at ~ 70?C for about 15 min.

F. Staining

    Stain with appropriate stains per individual protocol.

G. Coverslip o Dry on a hotplate at approximately 60C and add drop of mounting media. Place

    #1.5 coverslip over sections and examine.

    Instructions modified from original resin enclosure sheet and College of Vet Medicine, North Carolina State University (M. Dykstra)

Information below is from Electron Microscopy Sciences

JB-4 ? Embedding Kit

    JB-4 is water soluble embedding media which is based on Glycol Methacrylate (GMA) plastic embedding. It is intended for use in preparing samples for high resolution microscopes (HRLM). The catalyzed monomer acts as a dehydration and infiltration agent; therefore, complete dehydration through 100% ethanol is not necessary (although recommended for large or dense tissues). Conventional paraffin sections have a greater degree of shrinkage and produce inadequate morphology when compared to JB-4.

    Features:

    ; Thin sections (.5-2.0 micron) with excellent morphological structure preservation.

    ; Water-clear blocks; casts in 90 minutes max. at room temp

    ; Good lipid and enzyme retention when processing at low temperatures (4?C).

    ; Removal of JB-4 resin prior to staining is not necessary.

    ; Clearing agents such as xylene and chloroform are not needed.

    ; Higher clarity and contrast than with paraffin sections.

    ; Easy processing of difficult specimens such as calcified bone and delicate

    embryonic tissue.

    References:

    ; Hofman E.O. & Flores, T.R., High Resolution LM in Renal Path., Amer. J. of Clin.

    Path., 76:5 (1981)

    ; Beckstead, J.H., Blood, 57(6):1008 (1981)

    ; Brinn, N & Pickett, P., J. Histotech., 2(5):125-130 (1979)

    ; Block, Matthew H., et al., Lab. Med., 13(5)(1982)

    ; Helander, K.G., J. Microscopy, 132:223 (1983)

    ; Cole, M.B., J. Microscopy, 127:139 (1982)

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    ; Higuchi, S., et al., Stain Tech., 54(1):5-12 (1979)

    ; Van DeVeldt, S., Am. J. Clin. Path.,73:121 (1980)

    ; Horton, W.A., J. Histochem. Cytochem., 31:417 (1983)

     Tacha, D.E. & Richard, T.C.., J. Histotech., 4(2):59 (1981) ;

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