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TitleGenome visualization by classic methods in light microscopy
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Table of Contents
                            SERIES PREFACE
1.  Principles
2.  Tissue Preparation
3.  Staining
4.  Histochemical Methods
5.  Fluorescent Methods
6.  Observation Phases
7.  Preparation of Products
8.  Protocols
Examples of Staining Methods
Document Text Contents
Page 2


Page 105

Histochemical Methods


4.1.4 Visualization of Proteins

Among the methods used to visualize proteinic
parts, some are descriptive (HartigÐZachariasÕs
method, for instance) and others are actually
histochemical, such as original DanielliÕs tetra-
zoreaction method. Hartig–Zacharias’s method

This is a descriptive method, not a histochemical
one, but it permits one to visualize proteins spe-
❑ Fixative
All the classic Þxatives are convenient, but avoid
osmium tetroxide.
❑ Reagents

� Nuclear fast red
� Potassium ferrocyanide 2% in sodium

chloride 1%
� Ferric chloride 1% in distilled water

❑ Protocol
1. Dewax, hydrate.
2. Immerse in potassium ferrocyanide. 10 min
3. Wash with tap water.
4. Immerse in ferric chloride. 2 min
5. Wash with tap water.
6. Immerse in nuclear fast red. 1 min
7. Wash with tap water.
8. Dehydrate.
9. Mount.

❑ Results
Proteins are blue stained. Nucleic acids are red
(if nuclear fast red has been used).

➫See Chapter 7: Preparation of Products.

➫Ferric chloride is sometimes called Òiron

➫Washing between ferrocyanide and ferric
chloride is optional.

➫Nuclear fast red staining is facultative and
can be avoided to visualize nucleic acids.

➫This method is useful if quick results must
be obtained. Danielli’s tetrazoreaction ➫See Section T chloramine–Schiff method

This method is based on an oxidative deamina-
tion with a liberation of aldehyde groups. These
latter combine with SchiffÕs reagent to yield a
characteristic staining pattern.
❑ Fixative
All the classic Þxatives are convenient. Avoid
osmium tetroxide.
❑ Reagents

� SchiffÕs reagent Page 90 Thursday, October 5, 2000 10:37 AM

Page 106

4.1 General Methods


� PBS pH 7.5
—Disodium phosphate (14.198 g/L)

841 mL
—Monosodium phosphate (13.805 g/L)

159 mL
� T chloramine 1% in buffer
� Sodium thiosulfate (hyposulfite) 5 %
� Sulfurous water:
—Sodium metabisulfite 10% 10 mL
—Distilled water 190 mL

� Protocol
1. Dewax, collodion, hydrate.
2. Immerse in T chloramine. 6 h

at 37̊ C
3. Rinse quickly with distilled water.
4. Immerse in thiosulfate. 3 min
5. Rinse with distilled water.
6. Rinse with sulfurous water.
7. Wash with tap water.
8. Dehydrate.
9. Mount.

� Results
Proteins with terminal –NH2 groups are pinkish

� See Chapter 7: Preparation of Products.

� Collodion only if it is necessary.

� Thiosulfate eliminates excess of T chloram-
ine. Visualization of proteins by
Coomassie blue

� Fixative
All the classic fixatives are convenient.

� Reagents
� Triton X-100 1% in water

� Coomassie blue

� Protocol
1. Dewax, hydrate.
2. Immerse in Triton X-100. 15 min
3. Immerse in Coomassie blue. 45 min
4. Rinse with distilled water or PBS (pH 7.0).
5. Mount in aqueous medium.

� Coomassie blue is also called “light blue.”

� Triton X-100 is a detergent that makes phos-
pholipid layers permeable. It allows the dye to
penetrate. Its use is optional in certain cases.
� See Chapter 7: Preparation of Products.

� The action of Coomassie blue can be
increased to 60 min.
� It is possible to mount sections with glycerol
50% in distilled water.
� This method is classically used to visualize
all the proteins belonging to a tissue. Page 91 Thursday, October 5, 2000 10:37 AM

Page 209



Performic acid, clamping with, for puric and
pyrimidic bases, 87

Perfusion Þxation, 31Ð32
Periodic acid-silver diamine staining method,

Phloxin, preparation of, 148
Phosphomolybdic acid-G orange, preparation of,

Photonic microscope, 133Ð134

image formation in, 134
Picric acid Þxative, 28
Plastic wax embedding, 35Ð36

sectioning blocks from, 47, 162
Plastic wax sections, adhesion of, to slide, 50
Plastids, DNA of, 9
Prenant triple staining, 72Ð73
Progressive mode, of staining, 14
Prophase, 8
Propidium iodide staining method, 121
Protein detection methods, 90

Coomassie blue, 91
Danielli tetrazoreaction, 90
Hartig-Zacharias method, 90
T chloramine-Schiff method, 90Ð91

Puric and pyrimidic base detection methods
Caspersson spectrophotometric method, 85
clamping with dinitroßuorobenzene, 87Ð88
clamping with performic acid, 87
Danielli tetrazoreaction, 86Ð87
Danielli tetrazoreaction after benzoylation or

acetylation, 85Ð86
Pyronine, preparation of

Þrst formula, 152
second formula, 152

Pyronine staining, 15
Pyronine staining method, 94


Quantitative analysis, 19, 111
Quinacrine mustard staining method, 124Ð125


Radioactive actinomycin method, 111
Ramon y Cajal trichroma staining, 74
Reductive groups, staining, 17Ð18
Regaud hematoxylin, preparation of, 145
Resin embedding, 39Ð41
Resolution, microscopic, 134
Ribonuclease Brachet test, in basophilic staining, 96
Ribonucleoside, 9
Ribonucleotide, 9
Ribose, 9Ð10

Ribosomal RNA, 10
RNA, 9Ð10

extraction of, hydrochloric acid, 96Ð97
types of, 10

Romeis azan staining, 64
Romeis azan staining method, 164
rRNA, 10


Saffron, preparation of, 149
Salt formalin Þxative, preparation of, 142

cell cultures as, 25Ð26
tissue dissections as, 25
types of material used as, 25

Schiff reagent, preparation of, 152
Section preparation

adhesion to slide, 48
celloidin method, 50
gelatin method, 50
gelatinized slide method, 49
gelatinous water method, 49
glycerin-albumin method, 48Ð49
Maximow method, 50
water method, 48

block microtoming in, 43Ð44
block cutting prior to, 44
celloidin blocks, 47
difÞculties in, 44Ð46
embedded bone, 47
frozen samples, 47
parafÞn, parafÞn-celloidin, parafÞn-gelatin

blocks, 43Ð46, 161Ð162
plastic wax blocks, 47

celloidin protection on slides, 52
deparafÞning, and hydration, 51
Þxation of thin sections, 32Ð33
mounting, 131Ð135,

see also

Mounting sections


Fluorescent visualization methods;
Histochemical visualization methods;
speciÞc staining method

Silver methenamine, preparation of, 152
Silver methenamine staining method, 109Ð110
Small nuclear RNA, 10

deÞnition of, 52
dry blood, on lamella, 53
dry blood, on slide, 53
imprint, 52
nucleic acid staining methods for, 100Ð101
squash, 53
staining, 78Ð79
wet, 53

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Page 210



snRNA, 10


-Azure, preparation of, 152
Squash preparation, 53
Staining, 11Ð12,

see also

speciÞc staining method
acidophilic, 14
azan, 63Ð65, 65Ð66
azoic, 63Ð65
azure blue, 15, 17
basophilic, 14Ð15
carbonyl group, 18Ð19
Cleveland and Wolfe trichroma, 75
contrast, 68Ð80,

see also

Contrast staining
cresyl blue, 17
hematoxylin, 61Ð63
hematoxylin-eosin, 68Ð69
hematoxylin-phloxin, 69
hematoxylin-picro-indigo-carmine, 69Ð70
Herlant tetrachroma, 76
Mann-Dominici, 15
Masson trichroma, 70Ð72
Masson-Goldner trichroma, 72
May-GrŸnwald Giemsa, for smears, 78Ð79
metachromic, 15Ð17
methyl green, 15
nuclear, 59,

see also

Nuclear dyes
one-time trichroma, 74Ð75
Pappenheim panoptic, 78
paraldehyde fuchsin, 76Ð77
Prenant triple, 72Ð73
progressive mode, 14
quantitative analysis by, 19
Ramon y Cajal trichroma, 74
reductive group, 17Ð18
thin section, 66Ð68
thionin, 15, 17
toluidine blue, 15, 17
types of, 13Ð14
vessels for, 19


T chloramine-Schiff staining method, for proteins,

Telophase, 9
Thin sections

Þxation of, 32Ð33
staining, 66Ð68,

see also

Toluidine blue staining

Thionin, preparation of, 153
Thionin staining, 15, 17
Thionin-SO2 staining method, 107
Thymine, 5
Tissue preparation

cell culture, 54,

see also

Cell cultures
celloidin protection of, on slides, 52
deparafÞning, and hydration of, 51
dissection in, 25, 159Ð160
embedding in, 36Ð43, 160Ð161,

see also


Þxation in, 26Ð34,

see also


samples in, 25Ð26
sectioning in, 43Ð51,

see also

smears, 52Ð2Ð53,

see also

Toluidine blue, preparation of, 153
Toluidine blue staining, 15, 17

of thin sections, 66Ð67, 98
Toluidine blue-PAS staining, of thin sections, 67

method for, 166Ð167
Transfer RNA, 10
tRNA, 10
Trypsin, use of, 26
Turchini et al. staining method, pentose detection by,

2-D-ribofuranose, 9


Uracil, 10


Van Gieson picro fuchsin, preparation of, 149
Viral nucleic acids, 10




parafÞn entries


Z-DNA, 7
Zenker Þxative, preparation of, 143
Ziehl fuchsin, preparation of, 149

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