FIXATION and DEHYDRATION:
Tissue can be fixed with routine light microscopy fixatives. Best results are obtained with neutralized buffered formalins or Bouins. Specimen size should be kept small at 0.2 x 0.2cm.
Dehydrate samples through a graded series of ethanol 70%-95%.
Because the JB-4 resin is water soluble, complete dehydration through 100% ethanol is not necessary, although recommended especially for large or dense tissue. Clearing agents such as xylene or chloroform are not necessary.
Fixation, dehydration, and infiltration can be accomplished manually or automated with the use of a regular tissue processor used for paraffin processing. Processing through cold (4 degrees C) fixative, buffer rinse and infiltration resin can be used for optimal enzyme and antigen retention and preservation. This procedure uses the infiltration resin as the dehydrating agent replacing the alcohol series. No alcohol dehydration is needed, but recommended for large, bloody, or fatty tissue.
PREPARATION OF JB-4 CATALYZED INFILTRATION RESIN:
Prepare the infiltration resin as follows: 100ml of JB-4 Solution A add 0.90 grams of dry Catalyst C. Mix until dissolved. Careful weighing of the catalyst is necessary for correct polymerization control. This infiltration solution may be stored for 5-6 weeks at 4 degrees C in a dark bottle.
The percentage of catalyst added to Solution A should be decreased to 0.5%-0.7% when using large quantities for automatic processor units. This aids in solution preservation and minimizes heat sensitivity under processor conditions. Also, decreasing catalyst percentage (0.7%) seems useful in providing positive immunostaining. Infiltration time ranges from 2 hours to several days depending on size and tissue density. The tissue appears translucent and usually sinks to the bottom of the container. These solutions should always be kept cold. When processing for routine samples, infiltration solution should be changed 3-4 times, 30-90 minutes duration for each change. Solutions and tissue should be agitated on a rotator or hematology shaker during infiltration.
EMBEDDING:
Have embedding molds, labels, ice bath, gloves, instruments and cold fresh catalyzed Solution A ready before proceeding.
Prepare the embedding resin as follows: Add exactly 1ml of JB-4 Solution B to 25ml of fresh catalyzed Solution A. Never use exhausted infiltration resin for embedding. Stir well and place into an ice bath while embedding to retard premature polymerization. Anaerobic conditions are needed for polymerization.
Molds or embedding capsules must be filled and covered or capped tightly, using EBH-2 block holders Blocks will cut easier after removing from mold by exposure to air for a few hours. Polymerization is complete at room temperature in 50 minutes or less. NOTE: Polymerization proceeds more rapidly in larger batches; therefore, volumes should always remain under 50ml during polymerization. Polymerization will take somewhat longer in cold temperatures.
SECTIONING:
Optimal sectioning is performed with a microtome designed for plastic embedments. 0.5 micron-3 micron sections are cut with a dry glass or diamond knife, collected with forceps, and transferred onto a room temperature water bath surface, releasing sections before they touch the water. 1-2 drops of concentrated NH4OH added to the waterbath may aid in flattening sections. Sections are collected on pre-cleaned glass slides and air-dried before staining.
STAINING:
Dry sections are stained directly without xylene or alcohol pre-treatment. Longer staining times or higher stain concentrations may be necessary for thin sections. Alcohol or water rinses may be necessary after staining but the last step in the process should be water. Our Tissue-Tack (EMS Cat. #71301-01) may be used to affix tissue to the slide during lengthy procedures. Slides are mounted while still moist with most any mounting media.