Study of neuropathology has always been marked a certain mystique. This has been partly because of bewildering array of empirical staining techniques (Kiernan, 1999). For nuclear staining; mixtures of mordant dyes with appropriate metal slates are applied to sections of tissue. Hematoxylin can be classified according to mordant: alum, iron, tungsten, molybdenum, lead hematoxylin and without mordant. Also the most common iron hematoxylins are: Weigert, Heidenhain, Loyez and Verhoeff hematoxylin. In Weigert hematoxylin ferric chloride is used as the mordant/oxidant. The main use of Weigert hematoxylin is as a nuclear stain and a greater variety of structural details can be revealed by careful differentiation (Kiernan, 1999; Armed Forces Institute of Pathology, 1960).
Complexity of staining methods of Central Nervous System (CNS) come with complexity of (CNS). For that it is necessary for the researchers of neurosciences to have dyes that can be relied upon to give satisfactory and repeatable results without cost, time consuming and complexity, also it is more necessary for the researchers in growing country to have dyes that can be cost effectiveness in addition to satisfactory results. We investigated modification of iron weigert staining methods, both quantity (replacement of alum with iron) and quality (decreasing staining duration) for staining histoarchitecture of CNS (neurons) to introduce time, cost effectiveness and simplify method that can give satisfactory and repeatable results.
MATERIALS AND METHODS
Preparing tissues samples: For this work whole brain of Wistar-derived strain Adult male rats (12-13 week old and 200-250 g weight) collected. Routine processing for brain performed containing fixation in formaldehyde 10%, dehydrate in ascending ethanol, clearing by xylen, embedding in wax (paraffin) and cutting into 5-7 μm slides (7). Sections (from brain cortex, hippocampus, cerebellum) divided to three groups: one for staining by Hematoxyllin and eosin, second for staining by cresyl fast violet (that specially performed for Nisl substances in neuron) and last for staining by modification iron hematoxyllin methods. In cresyl fast violet method nissl substance, neurons and cell nuclei appeared purple dark blue.
Preparing staining chemicals: For our study we performed material and methods of Rozas iron hematoxyllin (Weigert 1904; Gray, 1954; Rozas, 1935) but different in some chemicals and procedure. We used aluminum nitrate 2 hydrate (AL (NO3)3.9 H2O as an alternative of ferric ammonium sulphate in routine Rozas method. Briefly this material used: Solution A: hematoxylin 0.6 g dye, aluminum nitrate 2 hydrate 1.5 to 2 g mordant, aluminum chloride 1.2 g mordant, distilled water 74 mL solvent 95% ethanol 6 mL solvent, glycerol 20 mL stabilizer. Solution B: aluminum nitrate 2 hydrates 20 g mordant, distilled water 100 mL solvent. For solution A, we dissolved the hematoxylin in the ethanol dissolved the ferric ammonium sulphate and the aluminum chloride in the water. Combined and then added the glycerol.
Procedure: Briefly this procedure performed: Bringing sections to water with xylene and ethanol. Slide in solution A for 10-20 min. This step is critical for this methods against Rozas iron hematoxyllin method we performed only 10-20 min for12-24 h in this step. Slide in solution B until differentiated. Washing well in running tap water to blue. Rinsing with distilled water. Counter staining, dehydration with ethanol, clear with xylene and mounting.
The results effects of the various staining methods on various areas of brain
in adult rats are shown in Fig. 1-5.
Modification of iron weigert stained nucleus of cells black. Also background of sections was cinereous. Timing of staining when compared with Rozas (1935) iron weigert method was ten times lower. Results of neurons and glia staining were different. Positive reaction to neurons and poor reaction to astrocytes appeared by performing this method.
Also results of staining In CA1 of hippocampus with population of pyramidal,
molecular, polymorph neurons was different from dentate gyrus with population
of astrocyte and neurons. In brain cortex only neurons has seen, while glia
have stained poorly. Detailed characteristics contain nucleus, nucleolus, nuclear
envelope and plasma membrane clearly appeared of purkinje and granular cells
of cerebellum cortex.
||Results staining cortex of cerebrum by three methods, A) Hematoxylin
and eosin, B) Modification iron hematoxylin, C) Cresyl fast violet
||Results staining C1 of hippocampus by two methods, A) Modification
iron hematoxylin, B) Cresyl fast violet
||Result staining dentate gyrus by three method, A) Hematoxylin
and eosin, B) Modification iron hematoxylin, and C) Cresyl fast violet
||Result staining cortex of cerebellum by three method, A) Hematoxylin
and eosin, B) Modification iron hematoxylin and C) Cresyl fast violet
||Results staining cortex of cerebellum by Modification iron
hematoxylin A) Spines of purkinje cells. B) Neuron with clearly visible
nucleus, nucleolus, nuclear envelop and C) Plasma membrane and glia surrounding
Also spines (axon) of purkinje cells clearly visible (Fig. 5).But
distinguish golgi cells in granular layer could not detected by this method.
Objective of the study were modification of iron weigert staining methods, both quantity (replacement of alum with iron) and quality (decreasing staining duration) for staining histoarchitecture of CNS (neurons) to introduce time, cost effectiveness and simplify method that can give satisfactory and repeatable results. Results of study support our objective, because this method is uncomplicated and time and cost value and can be used as an alternative method especially for neuron staining in histology and pathology departments.
Fortunately in study of neuropathology empirical techniques are being replaced
by reliable immunohistochemical methods. But for growing countries uses of these
methods are cost consuming. For nuclear staining, mixtures of mordant dyes with
appropriate metal slates are applied to sections of tissue. Hematoxylin solutions
can be classified according to which mordant is used: alum, iron, tungsten,
molybdenum, lead hematoxylin and without mordant (Kiernan, 1999). Also the most
common iron hematoxylins are: Weigert, Heidenhain,Loyez and Verhoeff hematoxylin.
In Weigert hematoxylin ferric chloride is used as the mordant/oxidant. The main
use of Weigert hematoxylin is as a nuclear stain and a greater variety of structural
details can be revealed by careful differentiation. In present study we replaced
iron mordant by alum mordant [aluminum nitrate 2 hydrate (AL (NO3)3.9
H2O] in weigert staining method. Alum hematoxylin staining may be
progressive or regressive. The former is usually preferred (Kiernan, 1999).
The binding of dyes to textiles has been studied for many years, but is still
not fully understood. Also documents related to modification of iron hematoxylin
and replacement of alum mordant by alum mordant is poor. For this reason we
could not compare our result with any pervious finding, because of different
study design that we performed. Little is known of the mechanism of nuclear
staining by iron-hematoxylin. Also little is known about the mechanism of modification
iron weigert. Horobin (1998) suggests that dye-metal complexes bound to chromatin
by both ionic and non-ionic forces. A number of dyes have been recommended as
substitutes for hematoxylin, particularly during the early to mid-1970s when
hematoxylin was in short supply. Although some closely mimic the features of
hematoxylin, none will serve as a complete replacement (Lillie et al.,
1973). We recommended time alteration to get better and detailed characteristics
of cells because staining time have different results.
Intact determination detailed characteristics contain nucleus, nucleolus, nuclear envelope and plasma membrane of neurons clearly appeared by modification of iron weigert staining methods. It was specifically responded in the nucleus neurons. Neither astrocyte responds to methods. Also cell spines (axons) in particular purkinje cells can be distinguished.