Table 4 Selected routine muscle stains and histochemical reactions (Dubowitz et al., 2020; Loughlin 1993; Udd et al. 2019)

1) Hematoxylin and eosin (HE)

General architecture, regeneration, necrosis, phagocytosis, perifascicular atrophy, nuclear internalization, rimmed vacuoles, and inflammation (Figs. 3 and 4).

2) Gomori modified trichrome:

Muscle fibers sarcoplasm is blue-green, nuclei are red, and collagen is light green (Figs. 3 and 4). Sarcoplasmic reticulum, mitochondria, tubular aggregates, cytoplasmic bodies and reducing bodies are dark red (Dubowitz et al., 2020; Loughlin 1993).

Congenital nemaline myopathy derives from the greek word “nema” that means “thread” (Dubowitz et al., 2020), that presents rod structures that may be observed in red on the Gomori modified trichrome stain (Fig. 4). On electron microscopy, these structure are electrondense rods with a lattice structure similar to the Z disks of the sarcomere (Fig. 6).

In mitochondrial myopathies, ragged red fibers, correspond to mitochondrial proliferation (Fig. 4).

3) Periodic acid Schiff (PAS) without and with diastase:

Glycogen, neutral mucopolysacarides, glycoproteins, mucoproteins, glycoproteins, glycolipids, some insaturated lipids and phopholipids (Figs. 3 and 4) (Dubowitz et al., 2020; Loughlin 1993). The evaluation of PAS and PAS with diastase (glycogen is diastase labile) stains is useful to the diagnosis of glycogenoses with intrasarcoplasmic glycogen accumulation (Fig. 4).

4) Oil-red-O or Sudan-black:

Neutral lipids stain red (Oil-red-O) (Figs. 3 and 4) or black (Sudan black).

5) Succinate dehydrogenase (SDH):

Enzymatic activity of the nuclear encoded mitochondrial respiratory chain complex II that reacts selectively with mitochondria (Figs. 3 and 4) (Dubowitz et al., 2020; Loughlin 1993). It is very useful for the diagnosis of mitochondrial myopathies.

6) Cytochrome-c-oxidase (COX):

Enzymatic activity of the mitochondrial encoded respiratory chain complex IV that reacts selectively with mitochondria (Figs. 3 and 4) (Dubowitz et al., 2020; Loughlin 1993).

7) Combined Cytochrome-c-oxidase/ succinate dehydrogenase (double COX-SDH):

Histochemical reactions performed in sequence on the same frozen section that permits the visualization of COX negative fibers with retention of SDH activity, useful for the diagnosis of mitochondrial myopathies (Fig. 4).

8) Nicotinamide adenine dinucleotide (NADH):

Oxidative enzyme on mitochondrial and endoplasmic reticulum, that is located in the sarcoplasm. It is very useful to demonstrate the muscle fiber intermyofibrillar network (Figs. 3 and 4) (Dubowitz et al., 2020; Loughlin 1993).

Congenital central core myopathy presents clear or empty areas on oxidative reactions COX, SDH, and NADH (Figs. 1 and 4). The name central core was given to this round structure that may sometimes occupy the central part of the muscle fibers and they may be visualized on transversal a longitudinal section (Figs. 3 and 4) (Dubowitz et al., 2020). However, these structure may present either central or peripheral disposition. On electron microscopy, these structures correspond to areas of myofibrillar disorganization with absence of scarcity of mitochondria and sarcoplasmic reticulum, that are responsible for the oxidative reactions (Figs. 1 and 6) (Dubowitz et al., 2020).

9) Myosinic ATPase:

Differentiation of muscle fiber types. In normal muscle, muscle fibers types are alternately organized in a checkerboard pattern (Figs. 3 and 4) (Dubowitz et al., 2020; Loughlin 1993). The type of the fiber is determined by the motor neuron that innervates each fiber and varies in each muscle (Table 3). This reaction if useful to demonstrate type grouping in neurogenic muscle abnormalities (Fig. 4). Type 1 fiber predominance is common in congenital myopathies. In congenital fiber type disproportion, there is type 1 fiber predominance and atrophy.

10) Acid phosphatase:

Areas of increased lysosomal activity, necrotic fibers, and lipofuscin. It is very useful for the diagnosis of glycogenosis type II (Pompe disease) and some vacuolar myopathies (Dubowitz et al., 2020; Loughlin 1993).

11) Myophosphorylase:

Enzyme present in the intermyofibrillar space aqueous sarcoplasm (Loughlin 1993). Its deficiency is useful for the diagnosis of glycogenosis type V (McArdle disease) (Fig. 4) (Dubowitz et al., 2020).

12) Phosphofructokinase:

The reaction may be absent in patients with glycogenosis type VII (Tarui disease) (Dubowitz et al., 2020).

13) Myoadenylate deaminase:

The histochemical reaction that may be absent in some patients with exertional myalgia (Dubowitz et al., 2020).

14) Nonspecific esterase:

This stain highlights neuromuscular junctions, myotendinous junctions, phagocytosis, and small angulated denervated fibers (Dubowitz et al., 2020).

15) Alkaline phosphatase:

It highlights the normal blood vessels and it is increased in the perimysium in some inflammatory myopathies (Dubowitz et al., 2020).

16) Menadione-linked alpha-glycerophosphate:

This stain is very useful for the diagnosis of reducing body myopathy (Dubowitz et al., 2020).

17) Congo red:

This stain that permits the visualization of amyloid deposits (Dubowitz et al., 2020).