### Generic template - SUMMARY OF PROCEDURES FOR [NERVE BIOPSIES]{.underline}

# OPERATING ROOM PROTOCOL

> **Supplies/Reagents/Equipment Required in Operating Suite at
> Initiation of Procedure:**

-   3% Glutaraldehyde in 0.1M sodium cacodylate buffer

-   Muscle/Nerve Biopsy Clamps (Rayport Muscle Biopsy Clamp \# SU
    130-1112, Allegiance Healthcare Corp., Government Customer Service,
    3651 Birchwood Drive, Waukegan, Illinois 60085, Telephone
    800-444-1166) or Wooden Applicator Sticks

-   Zeus/Michel's Fixative for Immunofluorescence (Wampole Labs,
    Division of Carter-Wallace, PO Box 1001, Cranbury, NJ, Telephone
    800-257-9525, Ext 3; Catalog \# 0102)

-   Zeus/Michel's Washing Solution, see above, Catalog# 0103

> **Clamped Specimens for Light Microscopy and Electron Microscopy**

-   Expose nerve (generally sural nerve) by gentle dissection, with care
    taken not to traumatize the nerve

-   Clamp the nerve (or a single nerve fasicle) **in situ** with
    muscle/nerve biopsy clamp or suture the nerve **in situ** to a
    wooden applicator stick

-   Section nerve free at either end of clamp/stick and remove with
    nerve attached to clamp/stick

-   Place IMMEDIATELY into 3% Glutaraldehyde in buffer.

> Note: ***Fixation in glutaraldehyde after removal must be as rapid as
> possible***
>
> Glutaraldehyde fixation is continued overnight (or longer, if a
> weekend) in the refrigerator until the specimen in glutaraldehyde is
> packaged for shipping as described below. The specimen in
> glutaraldehyde must remain on the muscle clamp for at least 4 hours.
> At the end of the 4 hours, the tissue may be unclamped, but it still
> must remain in glutaraldehyde until further processing occurs. Label
> all containers with the patient\'s name, social security number, type
> of specimen and type of fixative in the container.
>
> **Unclamped Specimens for Immunofluorescence**

-   Expose nerve as above

-   Dissect segment (at least 1mm cube), remove any excess fat, and
    place in Michel's fixative at room temperature

-   Label outside of all containers with the patient\'s name, social
    security number, type of specimen, type of fixative in the container
    and the date/time the specimen is put into the fixative

-   Ship all tissues in immunofixative by the most rapid method
    possible, e.g. air express, overnight mail, etc., as the
    immunoreactivity declines with time in fixative.

> **PACKAGING AND SHIPPING TISSUE**
>
> Check that all containers are labeled with the patient\'s name and
> social security number and any other pertinent information. On the
> accompanying SF515, include information regarding the type of tissue,
> pertinent clinical history (including preoperative diagnosis if
> applicable), and who should be contacted with results (including phone
> and/or Pager ID numbers).
>
> Please also include a copy of any prior pertinent surgical pathology
> or electron microscopy reports (if available) and/ or comments
> concerning the questions to be addressed by electron microscopy. It is
> helpful if a representative H&E slide can be sent as well. (This slide
> will be returned.)
>
> Make sure all caps on vials are secure and seal with parafilm.
> Transport or mail all specimens to:
>
> **Electron Microscopy Laboratory, Room XXXX**
>
> **Pathology and Laboratory Medicine Service, 113**
>
> **Local VA Medical Center**
>
> **000 Local Street**
>
> **City, State Zip**
>
> Be sure to label package on outside as "URGENT: PATIENT SPECIMENS".
> Specimens obtained after hours or on weekends should be held in the
> appropriate fixatives and mailed on the next work day.
>
> Address questions concerning submission of tissues or other materials
> for electron microscopy to: **Local Names/contacts/addresses/phone
> numbers**

## NOTES:

1.  The nerve tissue fixed in glutaraldehyde as described above will be
    studied by light and electron microscopy. If indicated, an aliquot
    of this same sample can also be studied by nerve fiber teasing as
    well.

2.  Please note that the above protocol does not use formalin for any
    portion of the nerve biopsy. Rapid fixation in glutaraldehyde is the
    preferred method of preservation.

> **SUMMARY OF PROCEDURES PERFORMED FOR NERVE BIOPSIES**
>
> **ON SITE AT Local VAMC**

## VAMC ELECTRON MICROSCOPY PROTOCOL

> The nerve specimen is removed from the clamp, and rinsed in
> buffer**.** After the segments of nerve crushed by the clamp have been
> cut away and discarded, a single cross section is taken from each end,
> post-fixed in osmium and embedded in epon. Cross-sections, 1 m in
> thickness, are stained with toluidine blue for light microscopy. In
> selected cases thin sections will be cut for electron microscopy. The
> remaining portion of nerve may be dehydrated through graded alcohols
> into cedar wood oil for teased nerve preparation if indicated.
> (Individual myelinated axons are placed on a clean glass slide,
> partially dried, and coverslipped with permanent mounting medium.)

## VAMC IMMUNOFLUORESCENCE PROTOCOL

> Specimens are removed from Michel's fixative and washed in buffer
> (Michel/Zeus Washing Solution, \*see reagents). Excess buffer is
> removed with a blotter. The specimens are embedded in OCT medium with
> tissue cores parallel to the eventual plane of sectioning, then snap
> frozen in a cryostat with a heat sink or in liquid perfluohexane
> (-90C). Frozen sections are cut at 3 microns on a crystat and
> collected on coated slides (positively charged, e.g. Superfrost
> Plus™). The sections are stained by the direct immunofluorescence
> method using FITC-conjugated antibodies and coverslipped in aqueous
> mounting medium. The sections are viewed by a pathologist using an
> epifluorescence microscope possessing a mercury light source. The
> light is passed through an excitation filter, yielding a wavelength
> range of 470-510 nm, which causes the FITC label to fluoresce.