Paraffin Sectioning and Mounting Protocol

Glen MacDonald, January 6, 2000

The result of this procedure for mounting sections is to allow any section from a sample to be equated with its position in the sample tissue or relative to other sections in the series. In practice, this protocol is much easier to follow than it is to explain.

Sectioning:

A. Techniques.

Prepare paper strips for the ribbons. A photocopy master is on file to make pages of strips. Each strip has one end labeled for animal number and ribbon number. The direction of sectioning (A to P, or P to A) and section thickness are noted on the first ribbon and every subsequent tenth ribbon. A paper strip is attached by its unlabeled end to the knife or disposable knife holder by means of double sticky tape. The labeled end is towards you.

Section the ribbon onto a paper strip until the first section is approaching the labeled end of the strip. Now place the ribbon (on paper strip) in a ribbon box so that strips are oriented from left to right. That is, the first section cut from the block should be in the upper left corner with serial sections extending vertically. ALWAYS MAKE A SMALL "X" ON THE RIBBON STRIP WHERE A SECTION IS LOST AND MAKE A NOTE ON THE SECTIONING SHEET!

When you have filled the box with ribbons (with the first section of each ribbon at the labelled end of the strip, carefully cover the box. Always keep it horizontal, don't jar it or move it suddenly. Label the end of the box with the animal number, box number, section thickness and direction of sectioning on the label. The box number is relevant only if there is more than one box of ribbons for an animal.

 

B. Record keeping.

Put the date of sectioning on the RUBELAB HISTOLOGY SCHEDULE "Sectioned:_____..." Include the section thickness (e.g. 10 u, 6 u, etc.) and direction of sectioning (i.e. A to P, or P to A) on the same line. Record the total number of ribbons and how many ribbon boxes there are in the HISTOLOGY SCHEDULE.

 

 

 

 

 

Sections will always get lost during sectioning, but it is imperative that it be noted where the tissue was lost and how much was lost. In general, an "x" with no explanation means one section was lost. If more tissue than that is lost at any place in a ribbon, then a note should be made under sectioning comments. Multiple lost sections may be indicated by several x's, or a numeric notation such as "4x", on the strip.

Ribbon box example:


Mounting.

A. Preparation:

Slides - When ready to mount sections, have on hand enough subbed slides (refer to "Staining" section of this Procedure Manual). Label them on the frosted end with the animal number at the top for easy sorting. Every slide should have a slide number. The first slide, and every subsequent tenth slide should have the section thickness (10 , 6 , etc), series type (1/4, 1/8, etc) and sectioning direction (a p, or p a).

Water bath - The water bath should be rinsed out and refilled with fresh deionized water for mounting sections onto subbed slides. Wipe the surface of the water with a KimWipe to remove dust and debris. The water temperature should be about 43-45o C.

B. Technique:

Orientation - The sections should be placed on the slides in the order that they were sectioned, and thus removed from the ribbon. The orientation of sections on the slides will therefore match their orientation on the ribbon strips, i.e. A to P, with A at the labelled end of a strip corresponding to A at the label end of the slide.

Sub-serial mounting - Start mounting with the first ribbon in box #1, or at the beginning of the area of interest. If you are mounting a 1 in 4 series, mount section #4 as you count from the first section in the box, or from the first section in the selected subset of ribbons. If you are mounting every section, then cut off a piece of ribbon containing 4-6 sections (depending upon section size). Place the chain of sections on the water bath. When mounting sub-serial series, ring the sections around the water bath so that you can keep track of the A to P or P to A direction when mounting. Sub-serially collected sections may be linked together to form short ribbons. This way the sections for each slide can be kept under better control, with less chance for mixing sections.

Do not forget to include sections that have been lost during sectioning in your counting - these should be counted when marking a series of sections to be mounted. Treat sections similarly when damaged or lost while mounting.

Picking up the sections - Observe the sections floating on the water bath. The tissue will expand faster than the surrounding paraffin. The paraffin must be given time to expand or the mounted tissue will dry with wrinkles. When the section is lying flat, without appearing puckered, then pick it up on a slide. Pick up the sections before they start to disintegrate, otherwise you will be chasing bits of tissue and paraffin debris.

Place the unlabelled end of the slide into the water bath, holding the slide vertically. Move the sections with a brush or probe up to the slide and hold it gently in place while drawing the slide upwards out of the bath. The section will attach itself to the slide as the water bath meniscus drops relative to the section. Drain the slide on a paper towel and set upright in the wooden rack to dry. When done, place all of the slides in a staining rack and put on a slide warmer overnight. For immunocytochemistry, the slide warmer should be 40-45o C.

ALWAYS WRITE DOWN PRECISELY WHAT YOU DID ON THE HISTOLOGY SHEET.

C. Recordkeeping

Put the date on the sacrifice sheet under Mounting comments. Write down the direction of mounting and what sort of series is being mounted. By convention, the label end of the slide is given the same orientation as the label end of the ribbon strip. Also mark down any unusual mounting methods, subbing and cleaning methods for slides.

 

 

EXAMPLE MOUNTING RECORD

Ribbon

number

Number of sections

saved

per ribbon

Number of sections

taken

per ribbon

Section

mounted

number

Slide number

Section numbers each slide

1

33

8

1-8

1A

1a-4a

2

31

7

9-15

2A

5a-8a

 

 

 

 

3A

9a-12a

 

 

 

 

4A

14a,15a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

section13a

lost

 

 

 

 

 

 

 

Summary:

2 Ribbons TOTAL

64 sections TOTAL

1 Box TOTAL

15 sections were saved, 14 were mounted on 4 slides of series "A"

section 13 was lost

The columns are filled in for each series if more than one series is being mounted from the same ribbons.

"Ribbon number" - identifies each ribbon from which sections are taken.

"Number of Sections saved" - is the total number of sections in each ribbon. It is most easily determined by counting in multiples of the sections marked for mounting for the first series.

"Number of Sections taken" - states how many sections were taken from each ribbon for any mounting series. This information is marked down on the same line as the ribbon number, next to the "number of sections saved" for that ribbon.

"Section mounted number" - each section mounted has a number corresponding to its place in a series of mounted sections (e.g. if 4 sections are mounted from ribbon 1, they are "section mounted number" 1-4; if 6 sections were mounted from the next ribbon, Ribbon 2, they would be "section mounted number" 5-10; if 5 sections were mounted from ribbon 3, they would be "section mounted number" 11-15; and so on for all the ribbons from which sections were mounted). If sections were mounted from ribbons 6-19 only, then the "section mounted number" would begin with "section mounted number" 1 as the first section mounted from ribbon number 6

Summary - Each section on a slide will have a "section mounted number" which it will keep forever. This matches its position both on the slides and among the sections taken from the ribbons. As you mount the tissue, write down each slide number and what sections it contains (i.e. slide #1, 1-4; slide #2, 5-8; and so) on for every slide. The number of sections taken from the ribbons should always match the number mounted on the slides, plus or minus 1 or 2 sections at most.

ALWAYS MAKE A NOTE OF TISSUE LOST DURING MOUNTING, TRY TO KEEP YOUR SERIES INTACT BY ACCOUNTING FOR ALL SECTIONS WHEN SELECTING SECTIONS TO BE MOUNTED.

IT IS VITAL TO WRITE DOWN EVERYTHING YOU DID AS YOU MOUNT OR IMMEDIATELY UPON COMPLETION OF EACH MOUNTING SERIES.

Multiple mounting series.

When mounting more that one series from any set of ribbons, the counting for the next series is begun with the section immediately before the first section of the preceding series. This is to ensure that the first few sections of an animal will not be ignored. In other words, if counting began at the first section of a ribbon, then begin counting the new series with the last section of the preceding ribbon. If there is no preceding ribbon to that animal, proceed as though it existed.

Each successive mounting series for an animal is designated by a capital letter, starting with "A" for the second mounting series. The mounted sections would then be given a small case "a" appended to the "section mounted number".

Mounting for autoradiography.

Use the appropriate slides for autoradiography to prevent background grains. Mount sections such that a .5 cm margin is left between the sections and the slide label and the slide end. The margins will ensure that the first section is fully immersed in the emulsion and that the last section is not obscured by emulsion build-up while the slides are draining.

nfocal files, this may be useful for situations when the sample has drifted during a z-series or time lapse acquisition.