MDC1A_M.1.2.003
Page 1 of 9 Plea quote the u of this SOP in your Methods. Histopathology in Masson Trichrome stained muscle ctions SOP (ID) Number MDC1A_M.1.2.003
Version 1.0
Issued April 19th, 2011
Last reviewed January 28th, 2014
Authors Markus A. Rüegg & Sarina Meinen (Biozentrum,
University of Bal, Bal, Switzerland)
Working group
members
Valerie Allamand (Inrm, U974, Institut de Myologie,
Paris, France)
Jeffrey B. Miller (Neuromuscular Biology & Dia
Group, Boston University School of Medicine, USA)
Madeleine Durbeej (Faculty of Medicine, Lund
西米的做法
University, Lund, Sweden)
Dean J. Burkin (Department of Pharmacology,
University of Nevada, Reno, NV, USA)脸起皮是怎么回事
接杀Anne M. Connolly (University of Washington School of
Medicine, St Louis, MO, USA)步出夏门行
Janice Dominov (University of Massachutts Medical
School, Worcester, MA, USA)
SOP responsibles Markus A. Rüegg, Sarina Meinen
Official reviewer Valerie Allamand
MDC1A_M.1.2.003
TABLE OF CONTENTS
1. OBJECTIVE (3)
2. SCOPE AND APPLICABILITY (3)
3. CAUTIONS (3)
4. MATERIALS (3)
5. METHODS (4)
5.1 Embedding of muscle (4)
5.2 Cryoctioning (5)
5.3 Masson TrichromeStaining (5)
5.4 Image analysis and quantification (6)
5.4.1 Image acquisition (6)
5.4.2 Manual Quantifications (7)
6. EVALUATION AND INTERPRETATION OF RESULTS (7)
7. REFERENCES (8)
8. APPENDIX (9)
MDC1A_M.1.2.003
1OBJECTIVE
This document describes a method and provides reference images for the histological characterisation of dystrophic muscle from dy W/dy W mice.
2SCOPE AND APPLICABILITY
‘‘Trichrome’’ stains (Masson) are ud for distinguishing collagen from muscle tissue. In general, they consist of nuclear, collagenous and cytoplasmic dyes in mordants such as phosphotungstic or phosphomolybdic acid.
The replacement of muscle with fibrotic tissue is a hallmark of muscles from MDC1A patients and m
ou models. Since The Masson Trichrome staining procedure stains the collagen-rich fibrotic regions in blue, it is especially suited to asss and visualize the extent of fibrosis in dystrophic skeletal muscle on transver muscle ctions.
In addition and similar as with the H&E staining, Masson Trichrome stained ctions also reveal adipo tissue, the variation in muscle fiber diameters, the prence of small or rounded fibers (de-/regeneration) as well as centralized nuclei (indicative of regeneration). 3CAUTIONS
As with all histological staining, great care must be taken in preparation of the samples: ∙Accurate removal of the muscle(s) from the mou will ensure that the appropriate portion of the appropriate muscle is actually being consistently analyd.
∙Fixation (or freezing) should be done carefully to minimize shrinkage of tissues (or ice-artifact). It is important to identify areas of ice-artifact in frozen ctions and to
distinguish this from actual areas of muscle necrosis.
∙Accurate embedding is esntial as all ctions must be embedded the same way.
∙Bad cutting (e.g. a blunt blade which leaves knife scores in the ction) or folding of the muscle ct
ion can make it hard to analy a ction accurately.
4MATERIALS
▪Cork plates (cut into quadrates of approx. 1.5cm x 1.5cm)
▪Isopentane: Merck 1060561000 or Sigma-Aldrich 27,034-2
▪Gum Tragacanth: Sigma-Aldrich (Cat. No. G1128)
MDC1A_M.1.2.003
▪Tissue-Tek O.C.T: Sakura (Cat. No. 4583)
▪Superfrost Plus Slides: Millian (SFPlus-42) or ThermoScientific Menzel Glär (J1800AMNZ)
▪PapPen Liquid Blocker (NANDAI Trading, Japan)
▪Paraformaldehyde (PFA): Fluka (Cat. No. 76240) or 4% Paraformaldehyde (Sigma P6148)
▪Bouin's solution (Sigma HT10132)
▪Weigert’s Iron Hematoxylin Solution Part A & Part B (Sigma-Aldrich, HT107 & HT109) ▪Accustain Trichrome Stain (Masson) Kit (Sigma-Aldrich, HT15)
生活污水处理工艺▪Glacial Acetic Acid (Merck 100063.1000)
▪100% ethanol, 90% ethanol, 70% ethanol
▪Xylene
▪Xylene-bad mounting media: DePeX mountant (BDH, 361254D)
Stock solutions:
100ml of 4% neutralized PFA: Heat ~50ml ddH2O to 60°C and add 4g of PFA while stirring (in a hood!). Then add 1M NaOH until PFA is dissolving (pH= 7.4-7.5). Fill up to 100ml with 2xPBS (end conc. 1x). Filter the PFA solution and check pH 7.4-7.5.
5METHODS
5.1Embedding of muscle
After disction, the muscles (e.g. Diaphragm, Triceps, Quadriceps, TA, EDL, Gastrocnemius, etc) are mounted on a small mound of 10% Gum Tragacanth that is placed on a cork disc. For bigger muscles (Triceps, Quadriceps, TA) prepare a little hole in the gum tragacanth and stick the muscle into it. Smaller muscles (Soleus, EDL) are looped around the forceps, stuck into the Gum and then stretched by pulling out the forceps. Ensure that the muscles are totally covered by the gum and that they are placed with the distal end of the muscle facing down and the proximal side up. Alternatively, muscles can be embedded in Tissue-Tek O.C.T. freezing medium in cryomolds (e.g Tissue-Tek 4565 or 4566). U the minimum amount of O.C.T. possible to cover the muscles, thus allowing rapid freezing to occur.
Place a cold-resistant beaker of isopentane into liquid nitrogen and allow cooling to -150°C. When the correct temperature is attained ‘sludge’ will app ear in the bottom of the isopentane. Freeze the embedded muscle by placing it into the cooled isopentane for 20-40
MDC1A_M.1.2.003
冷不丁的意思
conds (longer contact times can result in the formation of cracks in the samples; insufficient time c
an result in freezing artifacts) and then transfer the muscle sample to dry ice. For short-term or long-term storage keep the samples in -20°C or -80°C freezer, respectively.
5.2Cryoctioning
开放的家庭To achieve a thermal equilibration before cryoctioning, store the samples overnight in the -20°C freezer and place them into the cryostat for at least 20 minutes before further processing. Mount the sample on the round metallic mount of the cryostat with Tissue-Tek O.C.T. The knife should be pre-cooled to -20°C and the muscle sample to -22°C. Make 7-12 µm-thick ctions and collect them on warm (RT) Superfrost Plus slides. Let the ctions dry at RT for 1hr and then store the unstained slides at -20°C.
5.3Masson Trichrome Staining
Weigert’s iron hematoxylin stains the nuclei in black, Biebrich scarlet-acid fuchsin stains cytoplasm & muscle fibers in red and after treatment with phosphotungstic and phosphomolybdic acid, collagen is stained in blue with aniline blue.
Staining procedure:
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1.Preparation: Bring the slides to room temperature and surround the ctions with a
liquid blocker (PapPen) to prevent running away of the incubation solutions.
2.1° Fixation: Fix the cryoctions in 4% PFA or in 10% Formalin at RT for 1hr in the
hood.
3.2° Fixation: Re-fix ctions in Bouin's solution at RT overnight to intensify the colors
and increa the contrast between the tissue components.
4.Washing: Wash slides for 1-2 minutes under running tap water (18–26°C) to remove
yellow color from ctions. Then rin briefly in deionized/distilled water (ddH2O). 5.Weigert’s Hematoxilin Staining: Mix equal parts of Hematoxylin Solution A and
Solution B (stable for 10 days). Incubate the ctions for 5 minutes with Weigert’s
Iron Hematoxylin Solution to stain the nuclei dark. Discard the Hematoxilin solution.
6.Blueing: Put the slides in a glass chamber and wash under warm running tap water
for ~10 minutes to remove excess of Hematoxylin and to intensify the black color.
Rin 1 minute in ddH2O.
7.Cytoplasm Staining: Incubate the ctions for 5 minutes with Biebrich Scarlet-Acid
Fuchsin Solution to stain the fibers red. Discard the Solution.
