Lowicryl Tissue Processing

Pendrin IC cells

Pendrin in nonA nonB Intercalated Cells (Verlander et al., AJP Renal 2006)

Introduction

Transmission Electron Microscopy (TEM) is the method used to obtain high-resolution images of plant and animal tissue, allowing morphometric analyses of the internal detail of sectioned cells or direct preps of macromolecules, localizing where substances reside within a cell, or immunologically marking specific antigens in a cell. There are two basic types of electron microscopy: transmission electron microscopy (TEM) yields high-resolution ultrastructural detail from a transmitted electron beam through the tissue; and scanning electron microscopy (SEM) yields high-resolution surface morphology from a secondary electron beam.

Different steps in the preparation of samples for TEM usually include tissue fixation, dehydration, embedding, sectioning, and staining, procedures that take several days to complete. Fixation, an important step that basically kills the cells and “locks” in place the cellular components, may take place before or after the tissue is removed from the plant or animal subject, and most often utilizes an aldehyde as the fixation agent. As a general rule, samples that are intended for immunolocalization should initially be fixed in a very low concentration of glutaraldehyde, preferably 0.25 to 0.5%, to maintain antigenicity. Mixtures of glutaraldehyde with paraformaldehyde are commonly used since paraformaldehyde does not seem to interfere with antigenicity.

Embedding the tissue involves the removal of moisture (dehydration) using increasing concentrations of ethanol or acetone and subsequent infiltration with a formulated resin. Another obstacle to the immune response is the embedding media; acrylic resins usually work well, while epoxy resins are resistant and seldom show good results. Two alternatives would be either to do the immunolocalization steps on the fixed tissue before embedding in an epoxy (pre-embedding) or to avoid the use of resin and aldehydes altogether, using unfixed frozen samples that have been immersed in a cryoprotectant.

In this tutorial, we will use an acrylic resin known as Lowicryl® K4M (Polysciences, Inc. Warrington, PA). Lowicryl® K4M is processed in a hood and polymerized at -20oC under 360nm UV irradiation. It is highly sensitive to oxygen, so care must be taken to eliminate oxygen during polymerization by bubbling with dry nitrogen and tightly sealing the embedding mold. Extreme safeguards should be used when handling Lowicryl®, especially by skin sensitive people. Even a double latex-type glove will not prevent Lowicryl® from badly damaging sensitive skin. We recommend wearing a polyethylene glove underneath a latex glove and changing gloves frequently since it takes about 30 minutes. For Lowicryl® to penetrate through the polyethylene glove. Furthermore, Lowicryl® fumes or vapors are hazardous and a high-velocity fume-hood should be used to prevent facial skin problems.

Materials

  •             Tissue sample, fixed (submitted by PI in fixative or buffer)
  •             Buffer compatible with fixative used
  •             Small tissue forceps
  •             Sharp (double edge) razor blade
  •             Dental wax (Ted Pella, Inc., Redding, CA)
  •             Small labeled glass vials, one for each sample
  •             Embedding capsules, e.g. BEEM capsules (Ladd Research Industries, Williston, VT)
  •             0.1M NH4CL in PBS (134mg/25mL)
  •             Ethanols: 70%, 80%, 95%, 100%, chilled to 4oC
  •             Absolute Ethanol, freshly opened, RT
  •             Box with 360nm UV lights, to fit in freezer
  •             Lowicryl® K4M embedding kit (Polysciences, Inc.)

Protocol

CAUTION:  SAMPLES MUST NOT BE ALLOWED TO DRY OUT!  WHEN CHANGING SOLUTIONS, REMOVE AND REPLACE THEM IN ONE VIAL AT A TIME; LEAVE A SMALL AMOUNT IN THE BOTTOM OF THE VIAL; ADD NEXT SOLUTION AS QUICKLY AS POSSIBLE.

1. Refrigerate ethanols.

2. In a small drop of buffer on a square of dental wax, dissect tissue into 1mm3 pieces.

3. Transfer pieces into small labeled vials containing buffer.

4. Incubate 1mm3 tissue blocks in 0.1M NH4Cl in PBS for 1hr. @ 4oC.

5. Decant 0.1M NH4Cl and start dehydration procedure:

                        A. 70% Ethanol                                                          1 X 10 min. 4oC

                        B. 95% Ethanol                                                           1 X 10 min. 4oC

                        C. 100% Ethanol                                                         2 X 10 min. 4oC

                        D. 100% Ethanol        (freshly opened bottle)            2 X 10 min.  RT

CAUTION:  100% EtOH IS VERY VOLATILE AND WILL EVAPORATE ALMOST IMMEDIATELY:  CHANGE THESE SOLUTIONS AS QUICKLY AS POSSIBLE.

6. Mix K4M slowly, following kit instructions, without introducing air bubbles. Use a glass stir rod, or gently roll the sealed container to mix in the catalyst as it dissolves.

7.  Continue with resin infiltration:                                        (minimum)

                        E. 1:1 K4M and fresh 100% Ethanol             30 min.

                        F. 2:1 K4M and fresh 100% Ethanol              Overnight

                        G. 100% K4M                                                3 hrs. Min.

8.  Immediately before embedding, cover the container with parafilm, seal with tape, and (through a small hole in the parafilm) gently bubble the K4M with dry Nitrogen for 5 minutes. Keep it covered with the parafilm.

9. Removing the K4M with a disposable pipet through the hole, place a small amount of 100% K4M in labeled BEEM capsules and add tissue to the capsule, then completely fill the capsule to the top with K4M. Cap vials (allowing the least possible bubble at the top) and transfer to the polymerization box.

10.  Polymerize initially in UV-Light box in freezer                           24 hrs, UV light ON

11.  Then: in UV-Light box (UV ON) at RT under fume hood         2 to 3 days.

12.  Section sampling of blocks as soon as possible to determine that acrylic is satisfactorily polymerized.